ES2252019T3 - MORAXELLA CATARRHALIS BASB111 POLYPYPTIDE AND POLYPYPTIDE. - Google Patents

Abstract

An isolated polypeptide comprising an amino acid sequence that has at least 85% identity with the amino acid sequence of SEQ ID No. 2 over length

Description

BASB111 polypeptide and polynucleotide of Moraxella catarrhalis.

Field of the Invention

This invention relates to polynucleotides (referred to herein as "polynucleotide or BASB111 ") polynucleotides, to polypeptides encoded by them (referred to herein as "BASB111" or "polypeptide or BASB111 ") polypeptides, to materials and procedures of recombination for its production. In another aspect, the invention is refers to procedures for using these polypeptides and polynucleotides, including vaccines rente bacterial infections. In a further aspect, the invention relates to tests Diagnostics to detect infection by certain pathogens.

Background of the invention

Moraxella catarrhalis (also called Branhamella catarrhalis ) is a Gram-negative bacterium frequently isolated from the human upper respiratory tract. It is responsible for several pathologies, the main one being otitis media in children and young people and pneumonia in the elderly. It is also responsible for sinusitis, nosocomial infections and, less frequently, for invasive diseases.

Otitis media is an important disease in childhood as much for the number of cases as for its consequences potential. In the United States, more than 3.5 are registered each year million cases and it is estimated that 80% of children have experienced at least one episode of otitis before reaching the age of 3 years (Klein, JO (1994) Clin. Inf. Dis 19: 823). If left without treatment or becomes chronic, this disease can lead to a hearing loss that could be temporary (in the case fluid accumulation in the middle ear) or permanent (if damaged the auditory nerve). In babies, this hearing loss can Be responsible for a delay in speech learning.

Mainly, three species of bacteria are isolated from the middle ear of children with otitis media: Streptococcus pneumoniae , nontypable Haemophilus influenzae (NTHi) and M. catarrhalis . They are present in 60 to 90% of cases. The sequencing of the complete genome of H. influenzae is described in WO 96/33276. A review of recent studies shows that S. pneumoniae and NTHi both represent approximately 30% and M. catarrhalis approximately 15% of cases of otitis media (Murphy, TF (1996) Microbiol. Rev. 60: 267). Other bacteria can be isolated from the middle ear ( H. influenzae type B, S. pyogenes , etc.) but at a much lower frequency (2% of cases or less).

Epidemiological data indicate that, for pathogens found in the middle ear, colonization of the tract upper respiratory is an absolute prerequisite for the development of an otitis, however, others are also needed requirements to develop the disease (Dickinson, DP et al. (1988) J. Infect. Dis. 158: 205, Faden, HL et al. (1991) Ann. Otorhinol Laryngol 100: 612). These are important for trigger the migration of bacteria to the middle ear through Eustachian ducts, followed by the initiation of a process inflammatory. These factors are unknown to date. It has been postulated that a transient immune system abnormality that follow to a viral infection, for example, could cause a disability to control the colonization of the respiratory tract (Faden, HL and cabbage. (1994) J. Infect. Dis. 169: 1312). An alternative explanation is that exposure to environmental factors allows colonization more important in some children, who later become susceptible to developing otitis media due to the presence pathogen maintenance in the middle ear (Murphy, TF (1996) Microbiol Rev. 60: 267).

The immune response against M. catarrhalis is poorly characterized. The analysis of strains isolated sequentially from the nasopharynx of babies followed by 0 to 2 years of age, indicates that they frequently catch and eliminate new strains. This indicates that an effective immune response against this bacteria is prepared in colonized children (Faden, HL et al (1994) J. Infect. Dis. 169: 1312).

In the majority of adults tested, bactericidal antibodies have been identified (Chapman, AJ et al. (1985) J. Infect. Dis. 151: 878). Strains of M. catarrhalis show variations in their ability to resist serum bactericidal activity: in general, isolates from sick individuals are more resistant than those that are simply colonized (Hol, C et al. (1993) Lancet 341: 1281, Jordan, KL et al. (1990) Am. J. Med. 88 (supl. 5A): 28S). Therefore, serum resistance could be considered as a virulence factor of the bacteria. Opsonizing activity has been observed in the sera of children recovering from otitis media.

Human antigens have not been identified target of these different immune responses, with the exception of OMP B1, an 84 kDa protein whose expression is regulated by iron, and that is recognized by the sera of patients with pneumonia (Sethi, S, et al. (1995) Infect. Immun. 63: 1516), and of UspA1 and UspA2 (Chen D. et al. (1999), Infect. Immun. 67: 1310).

Some other membrane proteins present on the surface of M. catarrhalis have been characterized using biochemical methods or for their potential involvement in the induction of protective immunity (for review, see Murphy, TF (1996). Microbiol. Rev. 60: 267 ). In a mouse pneumonia model, the presence of antibodies produced against some of them (UspA, CopB) favors a faster clearance of lung infection. Another polypeptide (OMP CD) is highly conserved among strains of M. catarrhalis , and has homologies with a porse of Pseudomonas aeruginosa , which has proven effective against this bacterium in animal models. US-A-5,599,693 describes the cloning and expression of a 30, 80 and 100 kd outer membrane protein of M. catarrhalis and the potential utility of these proteins in vaccine preparation. US-A-5,607,849 describes the nucleotide sequence of protein E of the outer membrane of M. catarrhalis .

The frequency of Moraxella catarrhalis infections has risen dramatically in recent decades. This has been attributed to the emergence of multiplied antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer rare to isolate strains of Moraxella catarrhalis that are resistant to some or all conventional antibiotics. This phenomenon has created an inadequate medical need and a demand for new antimicrobial agents, vaccines, drug selection procedures and diagnostic tests for this organism.

Summary of the Invention

The present invention relates to BASB111, in particular BASB111 polypeptides and BASB111 polynucleotides, recombination materials and methods for their production. In another aspect, the invention relates to the use of these polypeptides and polynucleotides, including the prevention and treatment of Moraxella diseases. In a further aspect, the invention relates to diagnostic assays for detecting diseases associated with Moraxella infections and conditions associated with these infections, such as assays for detecting the expression or activity of BASB111 polynucleotides or polypeptides.

In more detail, according to one aspect of the present invention is provided an isolated polypeptide comprising a amino acid sequence that has at least 85% identity with the amino acid sequence of SEQ ID No. 2 over length full of SEQ ID No. 2.

In another aspect of the present invention, provides an isolated polypeptide of SEQ ID NO. 2.

In another aspect of the present invention, provides an immunogenic fragment of the polypeptide of the present invention in which (if necessary when attached to a vehicle) is capable of generating an immune response that recognizes the polypeptide of SEQ ID No. 2.

In another aspect of the present invention, provides an isolated polynucleotide encoding a polypeptide of The present invention.

In another aspect of the present invention, provides an isolated polynucleotide comprising a sequence of nucleotides encoding a polypeptide that has at least 85% of identity with the amino acid sequence of SEQ ID No. 2 on the full length of SEQ ID No. 2, or a nucleotide sequence complementary to said isolated polynucleotide.

In another aspect of the present invention, provides an isolated polynucleotide comprising a sequence of nucleotides that have at least 85% identity with a sequence of nucleotides encoding a polypeptide of SEQ ID NO. 2 on the complete coding region; or a nucleotide sequence complementary to said isolated polynucleotide.

In another aspect of the present invention, provides an isolated polynucleotide comprising a sequence of nucleotides that have at least 85% identity with SEQ ID NO. 1 over the full length of SEQ ID No. 1 or a sequence of nucleotides complementary to said isolated polynucleotide.

In another aspect of the present invention, provides an isolated polynucleotide comprising a sequence of nucleotide encoding the polypeptide of SEQ ID NO. 2.

In another aspect of the present invention, provides an isolated polynucleotide comprising the polynucleotide of SEQ ID No. 1.

In another aspect of the present invention, provides an isolated polynucleotide comprising a sequence of nucleotides encoding the polypeptide of SEQ ID No. 2, which can obtained by analyzing an appropriate library in conditions stringent hybridization with a labeled probe that has the sequence of SEQ ID No. 1 or a fragment thereof.

In another aspect of the present invention, provides an expression vector comprising a polynucleotide isolated from the present invention.

In another aspect of the present invention, provides a recombinant live microorganism comprising a expression vector of the present invention.

In another aspect of the present invention, provides a host cell comprising the vector of expression of the present invention.

In another aspect of the present invention, provides a membrane of a host cell of the present invention expressing an isolated polypeptide comprising a amino acid sequence that has at least 85% identity with the amino acid sequence of SEQ ID NO. 2.

In another aspect of the present invention, provides a method to produce a polypeptide that comprises an amino acid sequence that has at least 85% of identity with the amino acid sequence of SEQ ID No. 2, which comprises culturing a host cell of the present invention under sufficient conditions for the production of said polypeptide and recover the polypeptide from the culture medium.

In another aspect of the present invention, provides a procedure to express a polynucleotide of the present invention comprising transforming a host cell with the expression vector comprising at least one of said polynucleotides and culturing said host cell under conditions sufficient for the expression of any one of said polynucleotides

In another aspect of the present invention, provides a vaccine composition comprising an amount Effective of the polypeptide of the present invention and a carrier pharmaceutically acceptable.

In another aspect of the present invention, provides a vaccine composition comprising an amount Effective of the polynucleotide of the present invention and a vehicle pharmaceutically acceptable.

In another aspect of the present invention, provides an antibody generated against the polypeptide or the immunogenic fragment of the present invention.

In another aspect of the present invention, there is provided a method for diagnosing a Moraxella infection, which comprises identifying a polypeptide of the present invention, or an antibody that is immunospecific for said polypeptide, present in a biological sample of an animal suspected of having This infection

In still another aspect of the present invention, provides the use of a composition comprising an amount immunologically effective of a polypeptide or polynucleotide of the present invention in the preparation of a medicament for use in the generation of an immune response in an animal.

In a further aspect of the present invention, a therapeutic composition useful for treating humans with a Moraxella catarrhalis disease comprising at least one antibody directed against the polypeptide of the present invention and a pharmaceutically suitable carrier is provided.

Various changes and modifications within the spirit and scope of the described invention will be easily apparent to experts in the field from the reading of the following descriptions and from the reading of others parts of this description.

Description of the invention

The invention relates to BASB111 polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to BASB111 polypeptides and polynucleotides of Moraxella catarrhalis . The invention especially relates to BASB111 having the nucleotide and amino acid sequences presented in SEQ ID No. 1 and SEQ ID No. 2, respectively. It is understood that the sequences listed below in the Sequence Listing as "DNA" represent an example of an embodiment of the invention, since experts will recognize that these sequences can be used profitably in polynucleotides in general, including ribopolinucleotides.

Polypeptides

In one aspect of the invention, Moraxella catarrhalis polypeptides referred to herein as "BASB111" and "BASB111 polypeptides, as well as biological, diagnostic, prophylactic, clinical or therapeutically useful variants thereof and compositions comprising the same are provided.

The present invention further provides:

(a) an isolated polypeptide comprising a amino acid sequence that has at least 85% identity, preferably at least 90% identity, more preferably at minus 95% identity, most preferably at least the 97-99% exact identity with that of SEQ ID NO. 2;

(b) a polypeptide encoded by a isolated polynucleotide comprising a sequence of polynucleotides that have at least 85% identity, preferably at least 90%, more preferably at least the 95%, even more preferably at least 97-99% or exact identity with SEQ ID No. 1 over the full length of SEQ ID No. 1, or

(c) a polypeptide encoded by a isolated polynucleotide comprising a sequence of polynucleotides encoding a polypeptide that has at least the 85% identity, preferably at least 90%, more preferably at least 95%, even more preferably at minus 97-99% or exact identity with the sequence amino acid of SEQ ID NO. 2.

The BASB111 polypeptide provided in SEQ ID No. 2 is the BASB111 polypeptide of Moraxella catarrhalis strain MC2931 (ATCC 43617).

The invention also provides a fragment. immunogenic of a BASB111 polypeptide, that is, a portion contiguous of the BASB111 polypeptide having the same, or substantially the same immunogenic activity as the polypeptide comprising the amino acid sequence of SEQ ID NO. 2. This say, the fragment (if necessary when conjugated with a vehicle) is capable of generating an immune response that recognizes the BASB111 polypeptide. This immunogenic fragment may include, by example, the BASB111 polypeptide lacking a leader sequence N-terminal and / or a transmembrane domain and / or a C-terminal anchor domain. In one aspect preferred, the immunogenic fragment of BASB111 according to the invention substantially comprises the entire extracellular domain of a polypeptide having at least 85% identity, preferably at least 90% identity, most preferably at least 95% identity, more preferably at least 97-99% of identity with SEQ ID No. 2 over the full length of the ID SEC Nº 2.

A fragment is a polypeptide that has an amino acid sequence that is completely the same as one part but not the total of any amino acid sequence of any polypeptide of the invention. As with BASB111 polypeptides, the fragments may be "isolated" or comprised in a larger polypeptide which form a part or region, more preferably as a single continuous region in a single polypeptide.
higher.

Preferred fragments include, for example, truncation of polypeptides that have a portion of a sequence of amino acids of SEQ ID No. 2 or a variant thereof, such as a continuous series of remains that include a sequence of amino and / or carboxyl terminal amino acids. Are also preferred degradation forms of the polypeptides of the invention produced by or in a host cell. Fragments are also preferred characterized by structural or functional attributes such as fragments that comprise regions in alpha helix and that form a alpha helix, regions in beta sheet and forming a beta sheet, regions in turn and that form a turn, regions of turn and that they form turns, hydrophilic regions, hydrophobic regions, regions amphipathic alpha, unfriendly beta regions, flexible regions, regions that form surfaces, region that binds to the substrate and regions with high antigenic index.

Additional preferred fragments include a isolated polypeptide comprising an amino acid sequence that it has at least 15, 20, 30, 40, 50 or 100 contiguous amino acids of the amino acid sequence of SEQ ID No. 2 or an isolated polypeptide which comprises an amino acid sequence that has at least 15, 20, 30, 40, 50 or 100 contiguous truncated or deleted amino acids from the amino acid sequence of SEQ ID NO. 2.

The polypeptide fragments of the invention can be used for the production of the corresponding full length polypeptide by peptide synthesis; by therefore, these fragments can be used as intermediates for produce the full length polypeptides of the invention.

In particular, variants are preferred in the that have been replaced, deleted or added in any combination several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids.

The polypeptides, or immunogenic fragments, of the invention may be in the form of the "mature" protein or it can be a part of a major protein such as a precursor or a fusion protein It is often advantageous to include a sequence of additional amino acids containing secretory sequences or leaders, prosequences, sequences that help purification such as multiple histidine residues or an additional sequence for stability during recombinant production. Further, the addition of a polypeptide or lipid tail is also considered exogenous or polynucleotide sequences to increase the potential immunogenic of the final molecule.

In one aspect, the invention relates to soluble fusion proteins modified by genetic engineering, composed of a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of various immunoglobulins subclasses (IgG, IgM, IgA, IgE). The immunoglobulin is preferred as constant part of the heavy chain of human IgG, in particular IgG1, where the fusion takes place in the hinge region. In a particular embodiment, the Fc part can simply be removed incorporating a cleavage sequence that can be cleaved with the blood clotting factor Xa.

In addition, this invention relates to procedures for the preparation of these fusion proteins by genetic engineering and the use of these in the selection of drugs, diagnosis and therapy A further aspect of the invention is also refers to polynucleotides that encode these fusion proteins. Examples of fusion protein technology can be found in International Patent Applications No. W094 / 29458 and W094 / 22914.

Proteins can be chemically conjugated or express as recombinant fusion proteins to be produced in an expression system that allows the increase of levels compared to the unbound protein. The couple of fusion can help provide T cell epitopes collaborators (immunological fusion partner), preferably epitopes of collaborators recognized by humans, or help in the protein expression (increased expression) with yields greater than the native recombinant protein. Preferably, the fusion protein will be both an immunological fusion partner As a couple to increase expression.

Fusion partners include Haemophilus influenzae D protein and non-structural influenza virus protein, NS1 (hemagglutinin). Another fusion partner is the protein known as LytA. Preferably, the C-terminal portion of the molecule is used. LytA is derived from Streptococcus pneumoniae that synthesizes an N-acetyl-L-alanine amidase, LytA, (encoded by the lytA gene {Gene, 43 (1986) pages 265-272}), an autolysin that specifically degrades certain bonds in the skeleton of the peptide glycan. The C-terminal domain of the LytA protein is responsible for affinity for choline or for some choline analogue, such as DEAE. This property has been exploited for the development of plasmids expressing C-lytA in E. coli useful for the expression of fusion proteins. The purification of hybrid proteins containing the C-LytA fragment at its amino terminal end {Biotechnology: 10, (1992) pages 795-798} has been described. It is possible to use the repeated portion of the LytA molecule, which is located at the C-terminus that begins at residue 178, for example, residues 188-305.

The present invention also includes variants of the aforementioned polypeptides, which are polypeptides which vary with respect to the reference ones in substitutions of conservative amino acids, by which, a residue is replaced by another with similar characteristics. These typical substitutions they are between Ala, Val, Leu and Ile, between Ser and Thr, among the remains Asp and Glu acids, between Asn and Gln and between the basic residues Lys and Arg or the aromatic remains Phe and Tyr.

The polypeptides of the present invention can Prepare in any suitable way. These polypeptides include natural polypeptides, form-produced polypeptides recombinant, synthetically produced polypeptides or polypeptides produced by a combination of these procedures. The means for preparing these polypeptides are well understood in The technique.

It is more preferred that a polypeptide of the invention derives from Moraxella catarrhalis , however, it can preferably be obtained from other organisms of the same taxonomic genus. A polypeptide of the invention can also be obtained, for example, from organisms of the same family or taxonomic order.

Polynucleotides

It is an object of the invention to provide polynucleotides encoding BASB111 polypeptides, especially polynucleotides encoding the polypeptide of this document called BAS111.

In a particularly preferred embodiment of the invention, the polynucleotide comprises a region that encodes BASB111 polypeptides comprising a sequence shown in SEQ ID No. 1, which includes a full length gene or a variant of the same.

The BASB111 polynucleotide, provided in SEQ ID No. 1, is the BASB111 polynucleotide of Moraxella catarrhalis strain MC2931 (ATCC 43617).

A further aspect of the invention is to provide isolated nucleic acid molecules that encode and / or express BASB111 polypeptides and polynucleotides, especially BASB111 polypeptides and polynucleotides of Moraxella catarrhalis , including, for example, unprocessed RNA, ribozyme RNA, mRNA, cDNA, DNA Genomic and DNA B and Z. Additional embodiments of the invention include biologically, diagnostic, prophylactic, clinically or therapeutically useful polynucleotides and polypeptides, variants thereof and compositions comprising them.

Another aspect of the invention relates to isolated polynucleotides, including at least one complete gene, which encodes a BABS111 polypeptide that has a sequence of amino acids deduced from SEQ ID No. 2 and polynucleotides closely related to it and variants thereof.

In another particularly preferred embodiment of the invention a BASB111 polypeptide of Moraxella catarrhalis is contemplated which comprises or consists of an amino acid sequence of SEQ ID NO: 2 or a variant thereof.

Using the information provided herein, such as a sequence polynucleotide shown in SEQ ID No. 1, a polynucleotide of the invention encoding the BASB111 polypeptide of the invention can be obtained using conventional cloning and selection methods, such as cloning and Sequencing of chromosomal DNA fragments from bacteria using Moraxella catarrhalis Catlin cells as a starting material, followed by obtaining a full length clone. For example, obtaining a polynucleotide sequence of the invention, such as a polynucleotide sequence given in SEQ ID No. 1, typically, a library of chromosomal DNA clones of Moraxella catarrhalis Catlin in E. coli or in some other suitable hosts, it was incubated with a radioactively labeled oligonucleotide probe, preferably a 17-mer or greater, derived from a partial sequence. Next, clones bearing identical DNA to the probe can be distinguished using rigorous hybridization conditions. By sequencing the individual clones identified in this way by hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence, it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence. Conveniently, this sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described in Maniatis, T., Fritsch, EF and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) (see in particular, Hybridization Selection 1.90 and Molds for denatured double helix DNA sequencing 13.70). Direct sequencing of genomic DNA can also be performed to obtain a full length gene sequence. The polynucleotide shown in SEQ ID No. 1, illustrative of the invention, was discovered in a DNA library derived from Moraxella catarrhalis .

In addition, the DNA sequence shown in the SEQ ID No. 1 contains an open reading frame that encodes a protein which has approximately the number of amino acid residues shown in SEQ ID No. 2 with a deducted molecular weight that can be calculated using the molecular weight values of the remains of amino acids well known to those skilled in the art.

The polynucleotide of SEQ ID No. 1, between the codon starting at nucleotide number 1 and the termination codon that begins at nucleotide number 829 of SEQ ID No. 1, encodes the polypeptide of SEQ ID NO. 2.

In a further aspect, the present invention provides an isolated polynucleotide compound or consisting of:

(a) a polynucleotide sequence that has the less than 85% identity, preferably at least 90% of identity, more preferably at least 95% identity, even more preferably at least 97-99% or identity exact with SEQ ID No. 1 over the full length of SEQ ID No. 1, or

(b) a polynucleotide sequence that encodes a polypeptide that has at least 85% identity, preferably at least 90% identity, more preferably at minus 95% identity, even more preferably at least the 97-99% or 100% exact identity with the sequence of amino acids of SEQ ID No. 2, over the full length of the SEC ID No. 2.

A polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than Moraxella catarrhalis , can be obtained by a method comprising the steps of selecting an appropriate library under stringent hybridization conditions (for example, using a temperature in the range of 45-65 ° C and an SDS concentration of 0.1-1%) with a labeled or detectable probe consisting of or composed of the sequence of SEQ ID No. 1 or a fragment thereof and isolating a gene from full length and / or genomic clones containing said polynucleotide sequence.

The invention provides a full length identical polynucleotide sequence in a coding sequence (open reading frame) of SEQ ID No. 1. The invention also provides a coding sequence of a mature polypeptide or a fragment thereof, by itself, as well. as a sequence encoding a mature polypeptide or a reading frame fragment with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre, pro or prepro-protein sequence. The polynucleotide of the invention may also contain at least one non-coding sequence, including, but not limited to, at least 5 'and 3' non-coding sequences, such as transcribed but not translated sequences, termination signals (such such as rho-dependent and independent termination signals), ribosome binding sites, Kozak sequences, mRNA stabilizing sequences, introns and polyadenylation signals. The polynucleotide sequence may also comprise an additional coding sequence that encodes additional amino acids. For example, they can encode a marker sequence that facilitates purification of the fused polypeptide. In certain embodiments of the invention, the marker sequence is a hexahistidine peptide such as that provided in the pQE vector (Qiagen, Inc.) and is described in Gentz et al., Proc. Natl. Acad. Sci., USA 86 : 821-824 (1989), or an HA peptide tag (Wilson et al., Cell 37 : 767 (1984), which may be useful for purifying polypeptide sequence fused thereto. The polynucleotides of the invention they also include, but are not limited to, polynucleotides that comprise a structural gene and its naturally associated sequences that control expression
gene.

The nucleotide sequence encoding the BASB111 polypeptide of SEQ ID No. 2 may be identical to the sequence encoding the polypeptide contained in nucleotides 1 to 828 of SEQ ID No. 1. Alternatively, it may be a sequence that, as a result of the redundancy (degeneration) of genetic code, also encode the polypeptide of SEQ ID NO. 2.

The term "polynucleotide encoding a polypeptide" as used herein, encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, in particular a bacterial polypeptide and, more particularly, a Moraxella catarrhalis BASB111 polypeptide that has an amino acid sequence shown in SEQ ID NO. 2. The expression also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (eg, polynucleotides interrupted by an integrated phage, an integrated insertion sequence, a sequence of an integrated vector, an integrated transposon sequence, or due to RNA editing or reorganization of the enomic DNA) together with additional regions that may also contain coding and / or non-coding sequences.

The invention also relates to variants of the polynucleotides described herein that encode variants of a polypeptide having an amino acid sequence deduced the SEQ ID NO. 2. Polynucleotide fragments of the invention, for example, to synthesize polynucleotides of full length of the invention.

Additional embodiments especially Preferred are polynucleotides encoding variants of BASB111, which have the amino acid sequence of the BASB111 polypeptide of the SEQ ID No. 2, in which they are substituted, modified, deleted and / or add several, some, from 5 to 10, from 1 to 5, from 1 to 3, 2, 1 or none amino acid residue in any combination. Especially Preferred among these are substitutions, additions and deletions silent that do not alter the properties and activities of the BASB111 polypeptide.

Additional preferred embodiments of the invention are polynucleotides that are at least 85% identical in its full length to a polynucleotide encoding the polypeptide BASB111 having an amino acid sequence shown in SEQ ID No. 2, and polynucleotides that are complementary to these polynucleotides Alternatively, the most preferred are polynucleotides comprising a region that is at least 90% identical in full length to a polynucleotide encoding the BASB111 polypeptide and complementary polynucleotides to these. TO in this regard, polynucleotides are especially preferred when minus 95% identical in its full length. Further, Those with at least 97% among those with at least 95% and among these, they are especially very preferred those with at least 98% and at least 99%, being the most preferred with at least 99%.

Preferred embodiments are polynucleotides. encoding polypeptides that retain substantially the same biological function or activity that the mature polypeptide encoded by a DNA of SEQ ID No. 1.

In accordance with certain preferred embodiments of this invention, hybridizing polynucleotides are provided, especially in stringent conditions, with sequences BASB111 polynucleotides, such as polynucleotides in ID SEC No. 1.

The invention further relates to polynucleotides that hybridize with the polynucleotide sequences provided in this document. In this regard, the invention relates to especially to polynucleotides that hybridize in rigorous coding with the polynucleotides described is this document. As used in this document, the expressions "rigorous conditions" and "stringent hybridization conditions" means hybridizations that occur only if there is at least 95% and preferably at minus 97% identity between sequences. An example Specific to rigorous hybridization conditions is incubation overnight at 42 ° C in a solution comprising: formamide at 50%, SSCx5 (150 mM NaCl, 15 mM trisodium citrate), 50 sodium phosphate mM (pH 7.6), Denhardt solution x5, 10% dextran sulfate and 20 micrograms / ml naked salmon sperm DNA denatured, followed by washing the hybridization support in SSCx0.1 at about 65 ° C. The hybridization conditions and Washing are well known and examples appear in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N. Y., (1989), especially Chapter 11 of this. Hybridization solution can also be used with the sequences polynucleotides provided in the invention.

The invention also provides a polynucleotide that comprises or comprises a polynucleotide sequence obtained by analysis of an appropriate library containing the complete gene of a polynucleotide sequence shown in SEQ ID No. 1 under stringent hybridization conditions with a probe that has the sequence of said polynucleotide sequence shown in the ID SEQ No. 1 or a fragment thereof; and isolate said sequence polynucleotide The fragments useful to get said polynucleotide includes, for example, probes and primers are described completely elsewhere in this document.

As discussed elsewhere in this document with respect to the polynucleotide assays of the invention, by For example, the polynucleotides of the invention can be used as hybridization probe for RNA, cDNA and genomic DNA to isolate cDNA full length and genomic clones encoding BASB111 and to isolate cDNA and genomic clones from other genes that has a high identity, especially high sequence identity with the BASB111 gene. Generally, these probes will comprise at least 15 nucleotide residues or base pairs. Preferably you are probes will have at least 30 nucleotide residues or base pairs and it can have at least 50 nucleotide residues or base pairs. The Especially preferred probes will have at least 20 residues nucleotides or base pairs and may have less than 30 residues nucleotides or base pairs.

A coding region of a gene can be isolated BASB111 by selection using a DNA sequence provided in SEQ ID No. 1 to synthesize an oligonucleotide probe. TO then, a labeled oligonucleotide is used that has a sequence complementary to the one gene of the invention to analyze a genomic cDNA, DNA or mRNA library to determine what Library members hybridize with the probe.

Various methods well known to those skilled in the art are available for obtaining full-length DNA or for extending short DNAs, for example, based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al., PNAS USA 85 : 8998-9002,1988). Recent modifications of the technique, illustrated with examples in Marathon ™ technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In Marathon ™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an "adapter" sequence linked to each end. The nucleic acid amplification (PCR) is then performed to amplify the 5 '"lost" end of the DNA using a combination of oligonucleotide primers specific to the gene and the adapter. Then, the PCR reaction is repeated using "nested" primers, that is, primers designed to hybridize within the amplified product (typically an adapter specific primer that also hybridizes at the 3 'end of the adapter sequence and a specific primer of the gene that also hybridizes at the 5 'end of the selected gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full length DNA can be constructed by directly joining the product with the existing DNA to obtain a complete sequence, or by performing a full length independent PCR using the information from the new sequence for 5 'primer design.

The polynucleotides and polypeptides of the invention, for example, as reagents and research materials for the discovery of treatments and diagnosis of diseases, especially human diseases, such as described below in this document in relation to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derived from a sequence of SEQ ID No. 1, can be used in the procedures of this document as describes, but preferably for PCR, to determine if polypeptides identified in this document are transcribed or not by in whole or in part in infected tissue bacteria. Is recognized that these sequences will also be useful in the diagnosis of phase and type of infection that the pathogen has achieved.

The invention also provides polynucleotides encoding a polypeptide that is the mature protein plus additional amino or carboxy terminal amino acids or amino acids within the mature polypeptide (when the mature form has more than one polypeptide chain, for example). These sequences may play a role in the processing of a precursor protein to a mature form, may allow the transport of the protein, may lengthen or shorten the half-life of the protein or may facilitate the manipulation of a protein for assay or production, among other things. As is generally the case in vivo , additional amino acids can be removed from the mature protein by cellular enzymes.

For each polynucleotide of the invention, provides a complementary polynucleotide thereof. It preferred that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary.

A precursor protein, which has a form mature of the fused polypeptide with one or more prosequences may be an inactive form of the polypeptide. When the prosequences, generally these inactive precursors are activated. Before activation, some or all of the prosequences Generally, these precursors are called proproteins

In addition to the conventional representations of nucleotides A, G, C, T / U, the term "N" can also be used to describe certain polynucleotides of the invention. "N" means that any of the four nucleotides of DNA or RNA at this specific position in the DNA or RNA sequence, except that it is preferred that N is not a nucleic acid that, taken in combination with adjacent nucleotide positions, when reading in the correct reading frame, it had the effect of generate a codon of premature termination in this framework of reading.

In synthesis, a polynucleotide of the invention can encode a mature protein, a mature protein plus a leader sequence (which can be referred to as a preprotein), a precursor of a mature protein that has one or more prosequences which are not the leader sequences of a preprotein or a preprotein, which is a precursor to a proprotein that has a leader sequence and one or more prosequences, which are generally eliminate during processing stages that produce forms active and mature polypeptide.

According to one aspect of the invention, provides the use of a polynucleotide of the invention for purposes therapeutic or prophylactic, in particular, immunization genetics.

The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method, such as direct injection of plasmid DNA into muscle (Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al. ., Hum Gene Ther . (1983) 4: 419), the DNA supply complexing with specific protein vehicles (Wu et al., J. Biol. Chem . (1989) 264: 16985), coprecipitation of phosphate DNA calcium (Benvenisty and Reshef, PNAS USA , (1986) 83: 9551), encapsulation of DNA in various forms of liposomes (Kaneda et al., Science (1989) 243: 375), particle bombardment (Tang et al. , Nature (1992) 356: 152, Einsenbraun et al., DNA Cell Biol (1993) 12: 791) and infection in vivo using cloned retroviral vectors (Seeger et al., PNAS USA (1984) 81: 5849).

Vectors, host cells, expression systems

The invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, to host cells that have been genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombination techniques. To produce these proteins can also be used translation systems free of cells using RNA derived from the DNA constructs of the invention.

Recombinant polypeptides of the present invention by methods well known to experts in the field from manipulated host cells genetically comprising expression systems. Therefore in an additional aspect, the present invention relates to systems of expression comprising a polynucleotide or polynucleotides of the present invention, to host cells that are manipulated genetically with these expression systems and to the production of polypeptides of the invention by recombination techniques.

For recombinant production of the polypeptides of the invention, host cells can be genetically engineered to incorporate polynucleotide expression systems of the invention or portions thereof. The introduction of a polynucleotide into the host cell can be accomplished by procedures described in many conventional laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY , (1986) and Sambrook, et al., MOLECULAR CLONING : A LABORATORY MANUAL , 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), such as transfection with calcium phosphate, transfection mediated by DEAE-dextran, transvection, microinjection, transfection mediated by cationic lipid, electroporation, transduction, charge during scraping, ballistic introduction and infection.

Representative examples of suitable hosts include bacterial cells, such as streptococci, staphylococci, enterococci, E. coli , streptomyces, cyanobacteria, Bacillus subtulis , Neisseria meningitidis and Moraxella catarrhalis cells ; fungal cells, such as yeast cell, Kluveromyces , Saccharomyces , a basiodiomycete, Candida albicans and Aspergillus ; insect cells, such as Drosophila S2 and Spodoptera Sf9 cells ; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells, such as gymnosperm or angiosperm cells.

A wide variety of expression systems can be used to produce the polypeptides of the invention. These vectors include, among others, vectors derived from chromosomes, episomals and viruses, for example, vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such such as baculovirus, papova virus, such as SV40, vaccinia virus, adenovirus avian smallpox virus, pseudorabies virus, picornavirus, retrovirus and alphavirus and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage gene elements, such as cosmids or phagemids. The expression system constructs may contain control regions that also regulate the originated expression. In general, in this regard any system or vector suitable for maintaining, propagating or expressing polynucleotides and / or expressing a polypeptide in a host can be used for the expression. The appropriate DNA sequence can be inserted into the expression system by any variety of well known and routine techniques, such as, for example, those shown in Sambrook et al., MOLECULAR CLONING, A MANUAL LABORATORY , ( supra ).

In recombinant expression systems in eukaryotes, appropriate secretion signals may be incorporated into the polypeptide expressed for the secretion of a translated protein in the light of the endoplasmic reticulum, in the periplasmic space or in the extracellular environment. These signals can be endogenous to polypeptide or may be heterologous signals.

The polypeptides of the present invention can recover and purify from cell cultures recombinants by well known procedures that include precipitation with ammonium sulfate or ethanol, acid extraction, anionic or cationic exchange chromatography, chromatography on phosphocellulose, hydrophobic interaction chromatography, chromatography of affinity, hydroxyapatite chromatography and chromatography in lectin More preferably, it is used for purification metal ion affinity chromatography (IMAC). They can well-known techniques for protein refolding are used to regenerate the active conformation when the polypeptide is denatures during intracellular synthesis, isolation and / or purification.

The expression system can also be a recombinant living microorganism, such as a virus or a bacterium. The gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and infection in vivo with this live vector will lead to the in vivo expression of the antigen and the induction of immune responses. The viruses and bacteria used for this purpose are, for example: smallpox virus (for example, vaccinia, avian smallpox, canarypox), alphavirus (Sindbis virus, Semliki forest virus, Venezuelan equine encephalomyelitis virus), adenovirus , adeno-associated virus, picornavirus (poliovirus, rhinovirus), herpesvirus (varicella zoster virus, etc.), Listeria , Salmonella , Shigella , Neisseria , BCG. These viruses and bacteria can be virulent or attenuated in various ways to obtain live vaccines. These live vaccines are also part of the invention.

Diagnostic, prognostic, serotyping and mutation tests

This invention also provides the use of BASB111 polynucleotides and polypeptides of the invention for use as diagnostic reagents. The detection of polynucleotides and / or BASB111 polypeptides in a eukaryotic, particularly a mammal, and especially a human, will provide a diagnostic procedure for the diagnosis of the disease, staging of the disease or response of an infectious organism to drugs. They can detect eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected of being infected with an organism that comprises the gene or protein BASB111, at the level of nucleic acid or amino acid by means of a diversity of well known techniques as well as by procedures provided in this document.

Polypeptides and polynucleotides can be obtained for prognosis, diagnosis or other analyzes from body materials of supposedly infected and / or infected individuals. They can be used directly for the detection of polynucleotides from any of these sources, particularly DNA or RNA, or can be enzymatically amplified using PCR or any other amplification technique prior to analysis. RNA, particularly mRNA, cDNA and genomic DNA can also be used in the same way. Using amplification, the characterization of the species and strains of the infectious or resident organism present in an individual can be made by analyzing the genotype of a polynucleotide selected from the organism. Deletions and insertions can be detected by a change in the size of the amplified product compared to a genotype of a reference sequence selected from a related organism, preferably from a different species of the same genus or a different strain of the same species. Mutation points can be identified by hybridizing the amplified DNA with labeled BASB111 polynucleotide sequences. Perfectly or significantly coincident sequences of imperfect or more significantly mismatched doubles can be distinguished by digestion with DNase or RNase, for DNA or RNA respectively, or by detection of differences in melting temperatures or renaturation kinetics. Differences in the polynucleotide sequence can also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels compared to a reference sequence. This can be done with or without denaturing agents. Polynucleotide differences can also be detected by direct sequencing of DNA or RNA. See, for example, Myers et al., Science , 230 : 1242 (1985). Sequence changes at specific locations can also be discovered by nuclease protection assays, such as RNases, and V1 and S1 protection assay or a chemical cleavage procedure. See, for example, Cotton et al., Proc. Natl. Acad. Sci., USA , 85 : 4397-4401 (1985).

In another embodiment, an oligonucleotide probe array comprising BASB111 nucleotide sequences or fragments thereof can be constructed to make an effective selection of, for example, genetic mutations, serotype, classification or taxonomic identification. Matrix technology procedures are well known, have general application and can be used to answer a variety of questions in molecular ethics that include gene expression, genetic linkage and genetic variability (see, for example, Chee et al., Science , 274 : 610 (1996)).

Thus, in another aspect, the present The invention relates to a diagnostic kit comprising:

(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID No. 1 or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO. 2 or a fragment of the same; or

(d) an antibody against a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO. 2.

It will be appreciated that any of these kits, (a), (b), (c) or (d) may contain a substantial component. This kit is will use to diagnose a disease or susceptibility to a disease, among others.

This invention also relates to the use of polynucleotides of the present invention as reagents of diagnosis. Detection of a mutated form of a polynucleotide of the invention, preferably, of SEQ ID No. 1, which is associated with a disease or pathogenicity, will provide a tool for diagnosis that can be added to, or defined, a diagnosis of a disease, a prognosis of the course of a disease, a determination of a stage of the disease or a susceptibility to a disease, which are the result of subexpression, overexpression, or altered expression of the polynucleotide. They can detect organisms, particularly infectious organisms, which they have mutations in this polynucleotide at the polynucleotide level by a variety of techniques, such as those described in some point of this document.

They can also be detected at the level of polynucleotide or polypeptide cells of an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and / or polypeptide of the invention for a variety of techniques, allowing, for example, serotyping. For example, RT-PCR can be used to detect mutations in the RNA It is particularly preferred to use RT-PCR together with automated detection systems, such as, for example, GeneScan PCR of RNA, cDNA or can also be used for the same purpose. Genomic DNA For example, PCR primers can be used. complementary to a polynucleotide encoding the polypeptide BASB111 to identify and analyze mutations.

The invention further provides primers with 1, 2, 3 or 4 nucleotides removed from the 5 'and / or 3' ends. These primers can be used to, among other things, amplify DNA and / or BASB111 RNA isolated from a sample derived from a individual, such as a body material. The primers can used to amplify an isolated polynucleotide from a infected individual, so that the polynucleotide then can undergo various techniques to elucidate the sequence polynucleotide Thus, mutations in the sequence polynucleotide can be detected and used to diagnose and / or predict the infection or its stage or course, or to serotype and / or classify the infectious agent.

The invention further provides a method for diagnosing infections caused by Moraxella catarrhalis , which comprises determining from a sample derived from an individual, such as a body material, a high level of polynucleotide expression having a sequence of SEQ ID No. 1. The increase or decrease in the expression of a BASB111 polynucleotide can be measured using any of the methods well known in the art for the quantification of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNAse protection, type transfer Northern, spectrometry and other hybridization procedures.

In addition, a diagnostic test of according to the invention to detect overexpression of the BASB111 polypeptide compared to control tissue samples normal to detect, for example, the presence of an infection. Test techniques that can be used to determine levels of a BASB111 polypeptide, in a sample derived from a host, such as body material, are well known to experts in The matter. These test procedures include radioimmunoassays, competitive binding assays, analysis of Western blot, type antibody assays "sandwich", antibody detection and type assays ELISA

The polynucleotides of the invention can be used as components of polynucleotide matrices, preferably high density matrices or grids. These high density matrices are particularly useful for diagnostic and prognostic purposes. For example, a set of spots comprising a different gene and, in addition, a polynucleotide or polynucleotides of the invention, can be used as a probe, such as using hybridization or amplification of nucleic acids, using a probe obtained or derived from a body sample, to determine the presence of a particular polynucleotide sequence or related sequence in an individual. This presence may indicate the presence of the pathogen Moraxella catarrhalis and may be useful to diagnose and / or predict a disease or the course of a disease. A grid comprising several variants of the polynucleotide sequence of SEQ ID No. 1 is preferred. A grid comprising several variants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID No. 2 is also preferred.

Antibodies

Polypeptides and polynucleotides can be used. of the invention or variants thereof, or cells expressing the same, as immunogens to produce antibodies immunospecific for these polypeptides or polynucleotides, respectively.

In certain preferred embodiments of the invention antibodies against polypeptides or BASB111 polynucleotides.

Antibodies generated against the polypeptides or polynucleotides of the invention can be obtained by administering the polypeptides and / or polynucleotides of the invention, or epitope-bearing fragments of either or both, analogs of either or both, or cells expressing either or both , to an animal, preferably non-human, using routine protocols. Any technique known in the art that provides antibodies produced by continuous cell line cultures can be used for the preparation of monoclonal antibodies. Examples include various techniques, such as those of Kohler, G. and Milstein, C., Nature 256 : 495-497 (1975); Kozbor et al., Immunology Today 4 : 72 (1983); Cole et al., P. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY , Alan R. Liss, Inc. (1985).

The antibody production techniques of single chain (US Patent No. 4,946,778) can be adapted for produce single chain antibodies against polypeptides or polynucleotides of this invention. Mice can also be used transgenic, or other organisms or animals, such as others mammals, to express humanized immunospecific antibodies against the polypeptides or polynucleotides of the invention.

\hbox{(1991)  Nature   352 : 628).}

Alternatively, bacteriophage display technology can be used to select antibody genes with binding activities to a polypeptide of the invention from repertoires of genes and PCR amplified from human lymphocytes selected for possessing anti-BASB111 or from virgin libraries. (McCafferty, et al., (1990), Nature 348 , 552-554; Marks, et al., (1992) Biotechnology 10 , 779-783). The affinity of these antibodies can also be improved, for example, by chain redistribution (Clackson et al.,

 ? (1991) Nature 352: 628).} 

The antibodies described above may be used to isolate or to define clones that express the polypeptides or polynucleotides of the invention, to purify the polypeptides or polynucleotides, for example, by chromatography of affinity

In this way, among others, they can be used antibodies to BASB111 polypeptide or polynucleotide BASB111 to treat infections, particularly infections bacterial

Polypeptide variants, which include antigenic, epitopic or immunologically equivalent variants, they form a particular aspect of this invention.

Preferably, the antibody or variant thereof is modified to make it less immunogenic in the individual. For example, if the individual is human, the antibody may be more preferably "humanized", when the region or regions determining complementarity of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example, as described in Jones and cabbage. (1986), Nature 321 , 522-525 or Tempest et al., (1991) Biotechnology 9 , 266-273.

Antagonists and agonists: Assays and molecules

The polypeptides and polynucleotides of the invention can also be used to assess the binding of small substrate and ligand molecules in, for example, cells, cell-free preparations, chemical libraries and mixtures of natural products. These substrates and ligands can be natural substrates and ligands or they can be structural or functional mimetics. See, for example, Coligan et al., Current Protocols in Immunology 1 (2) : Chapter 5 (1991).

The selection procedures can simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes that carry the polypeptide or polynucleotide, or to a fusion protein of the polypeptide by a marker directly or indirectly associated with the compound candidate. Alternatively, the selection procedure may involve competition with a marked competitor. In addition, these selection methods can check if the candidate compound gives place to a signal generated by the activation or inhibition of polypeptide or polynucleotide, using appropriate detection systems to cells containing the polypeptide or polynucleotides. In In general, activation inhibitors are tested in the presence of a known agonist and the effect on activation by the agonist in the presence of the candidate compound. Can be used constitutively active polypeptides and / or polypeptides and constitutively expressed polynucleotides in procedures of selection of inverse agonists or inhibitors, in the absence of a agonist or inhibitor, testing whether the candidate compound gives rise to inhibition of polypeptide or polynucleotide activation, As may be the case. In addition, the selection procedures they can simply understand the steps of mixing a compound candidate with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measure the activity of the BASB111 polypeptide and / or polynucleotide of the mix and compare the activity of the polypeptide and / or polynucleotide BASB111 mix with a pattern. Fusion proteins, such such as those made from Fc portions and the BASB111 polypeptide, as described in this document, they can also be used to high performance screening assays to identify polypeptide antagonists of the present invention, as well as of Phylogenetically and / or functionally related polypeptides (see D. Bennett et al., J Mol Recognition, 8: 52-58 (1995) and K. Johnson et al., J Biol Chem, 270 (16): 9459-9471 (1995)).

Polynucleotides, polypeptides and antibodies that bind and / or interact with a polypeptide of the present invention can also be used to configure methods of selection to detect the effect of compounds added in the mRNA and / or polypeptide production in cells. For example, you can build an ELISA type test to measure the levels of secreted or cell-associated polypeptide using antibodies monoclonal and polyclonal by conventional procedures known in the art. This can be used to discover agents that can inhibit or enhance polypeptide production (also denoting antagonist or agonist, respectively) to from properly manipulated cells or tissues.

The invention also provides compounds of a selection procedure to identify those that enhance (agonists) or block (antagonists) the action of polypeptides or BASB111 polynucleotides, particularly those compounds that They are bacteriostatic and / or bactericidal. The selection procedure It may involve high performance techniques. For example, the selection of agonists or antagonists, a reaction mixture synthetic, a cellular compartment, such as a membrane, cell wrap or cell wall, or a preparation of any of them, comprising the BASB111 polypeptide and a substrate or labeled ligand that is incubated in the presence or absence of a candidate molecule that can be an agonist or antagonist of BASB111. The ability of the candidate molecule to be an agonist or BASB111 polypeptide antagonist is reflected in the decrease in binding of the labeled ligand or decreased production of product from this substrate. The molecules that bind to free of charge, that is, without inducing the effects of the polypeptide BASB111, are the most likely to be good agonists. Agonists are molecules that bind well and, as can be the case, increase the production rate of the product from substrate, increase signal transduction or increase chemical channel activity. The detection of the rate or level of, as may be the case, product production from substrate, signal transduction or chemical channel activity They can be enhanced using an indicator system. The systems indicators that may be useful in this regard include but no limitations, colorimetric, marked substrate that becomes in product, an indicator gene that is responsible for changes in the BASB111 polynucleotide or polypeptide activity and assays of binding known in the art.

Another test example for BASB111 agonists It is a competitive trial that combines BASB111 and a potential agonist with BASB111 binding molecules, BASB111 binding molecules natural recombinants, substrates or ligands or substrates or ligands mimetics, under appropriate conditions for an inhibition test competitive. BASB111 can be marked, such as with radioactivity or a colorimetric compound, so that it can be determined with accuracy the number of BASB111 molecules attached to a molecule of union or converted into a product to assess the effectiveness of the potential antagonist

Potential antagonists include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and / or polypeptide of the invention and thereby inhibit or extinguish its activity or expression. Potential antagonists may also be small. organic molecules, a peptide, a polypeptide, such as a closely related protein or antibody that binds to same sites in a binding molecule, such as a molecule of union, without inducing the activities induced by BASB111, thus preventing the action or expression of polypeptides and / or BASB111 polynucleotides ruling out the binding of polypeptides and / or BASB111 polynucleotides.

Potential antagonists include a small molecule that occupies the polypeptide binding site, thus preventing the binding of cell binding molecules, so that normal biological activity is prevented. Examples of small molecules include, but are not limited to, small organic molecules, peptides or peptide-like molecules. Other potential antagonists include antisense molecules (see Okano, J. Neurochem. 56 : 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION , CRC Press, Boca Raton, FL (1988), for a description of these molecules). Preferred potential antagonists include compounds related to variants of BASB111.

In a further aspect, the present invention is refers to soluble engineering fusion proteins genetics composed of a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of various immunoglobulins subclasses (IgG, IgM, IgA, IgE). The immunoglobulin is preferred as constant part of the heavy chain of human IgG, in particular IgG1, where the fusion takes place in the hinge region. In a particular embodiment, the Fc part can simply be removed incorporating a cleavage sequence that can be cleaved with the blood clotting factor Xa. In addition, this invention is refers to procedures for the preparation of these proteins from fusion by genetic engineering and the use of these in the selection of drugs, diagnosis and therapy. An additional aspect of the invention also relates to polynucleotides encoding these fusion proteins Examples of technology can be found fusion proteins in international patent applications No. WO94 / 29458 and WO94 / 22914.

Each of the polynucleotide sequences provided in this document can be used in the discovery or development of antibacterial compounds. The encoded protein after expression it can be used as a target for the selection of antibacterial drugs Additionally, the polynucleotide sequences encoding amino regions terminals of the encoded protein or Shine-Dalgarno, or other sequences that facilitate the translation of the respective mRNA, to build sequences antisense to control sequence expression encoder of interest.

The invention also provides the use of polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic host, preferably mammalian, responsible for the sequelae of the infection. In particular, the molecules of the invention can be used: in the prevention of adhesion of bacteria, in particular gram positive bacteria and / or gram negative, to eukaryotic extracellular matrix proteins, preferably from mammals, in medical devices or to proteins of the extracellular matrix in wounds, to block adhesion bacterial between proteins of the eukaryotic extracellular matrix, preferably mammals, and bacterial BASB111 proteins that they mediate tissue damage and / or to block the normal progression of the pathogenesis in infections initiated for reasons other than implantation of medical devices or by other techniques Surgical

Still in accordance with another aspect of the invention, BASB111 agonists and antagonists are provided, preferably bacteriostatic or bactericidal agonists and antagonists.

The antagonists and agonists of the invention can be used, for example, to prevent, inhibit and / or treat diseases.

In a further aspect, the present invention is refers to mimotopes of the polypeptide of the invention. A mimotope is a peptide sequence, quite similar to the native peptide (sequentially or structurally), which is capable of being recognized by antibodies that recognize the native peptide or is capable of generating antibodies that recognize the native peptide when it binds to a adequate vehicle

Peptide mimotopes can be designed for a particular purpose by adding, deleting or replacing the chosen amino acids. In this way, the peptides can be modified in order to facilitate conjugation to a protein vehicle. For example, it may be desirable for some conjugation procedures to include a terminal cysteine. In addition, it may be desirable for peptides conjugated to a protein carrier to include a hydrophobic end distal to the conjugated end of the peptide, so that the free unconjugated end of the peptide remains associated with the surface of the carrier protein. Thus presenting the peptide in a conformation that most closely resembles that of the peptide as found in the context of the entire molecule. For example, the peptides can be altered to have an N-terminal cysteine and a hydrophobic C-terminal tail amidated. Alternatively, the addition or substitution of a D-stereoisomeric form of one or more of the amino acids can be performed to create a derivative conducive, for example, to potential stability of the
peptide

Alternatively, peptide mimotopes can be identified using antibodies that are capable of binding themselves to the polypeptides of the present invention using techniques such as phage display technology (EP 0 552 267B1). This technique generates a large number of peptide sequences that mimic the structure of native peptides and are therefore capable of binding to native anti-peptide antibodies, but may not necessarily share significant sequence homology with the polypeptide themselves.
native.

Vaccinations

Another aspect of the invention relates to a method for inducing an immune response in an individual, particularly a mammal, preferably humans, which comprises inoculating the individual with a BASB111 polynucleotide and / or polypeptide, or a fragment or variant thereof, suitable for produce an immune response of antibody and / or T cells to protect said individual from an infection, particularly bacterial infection and more particularly a Moraxella catarrhalis infection. Procedures are also provided where this immune response delays viral replication. Yet another aspect of the invention relates to a method for inducing the immune response in an individual, which comprises providing this individual with a nucleic acid vector, sequence or ribozyme to direct expression of the BASB111 polynucleotide and / or polypeptide, or a fragment. or a variant thereof to express the BASB111 polynucleotide and / or polypeptide or a fragment or variant thereof in vivo to induce an immune response, such as producing an immune response of antibody and / or T cells, including, for example , T cells that produce cytokines or cytotoxic T cells, to protect said individual, preferably a human, from a disease, whether that disease is established or not in the individual. An example of the administration of the gene is to boost it into the desired cell in the form of a particle coating or otherwise. This nucleic acid vector may comprise DNA, RNA, a ribozyme, a modified nucleic acid, a DNA / RNA hybrid, a DNA-protein complex or an RNA-protein complex.

A further aspect of the invention relates to an immunological composition that when introduced into a individual, preferably a human, able to have induced in him an immune response, induces an immune response in this individual against a BASB111 polynucleotide and / or polypeptide encoded therefrom, in which the composition comprises a BASB111 polynucleotide and / or polypeptide encoded therefrom and / or comprises DNA and / or RNA that encodes and expresses an antigen of said BASB111 polynucleotide or polypeptide encoded from this or another polypeptide of the invention. The immune response can be used therapeutically or prophylactically and can take the form of antibody immunity and / or cellular immunity, such as cellular immunity arising from CTL or CD4 + T cells.

A BASB111 polypeptide or fragment thereof can be fused with a coprotein or chemical moiety that may or may not produce antibodies on its own, but that is capable of stabilizing the first protein and producing a fused or modified protein that will have antigenic properties and / or immunogenic and, preferably, protective properties. Thus, the fused recombinant protein preferably further comprises an antigenic coprotein, such as Haemophilus influenzae lipoprotein D, Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large coprotein that solubilizes the protein. and that facilitates the production and purification of it. In addition, the coprotein can act as an adjuvant in the sense of providing a generalized stimulation of the body's immune system that receives the protein. The coprotein can bind to the amino terminus or the carboxyl terminus of the first protein.

In a vaccine composition according to invention, a polynucleotide and / or may be present in a vector BASB111 polypeptide or a fragment, a mimotope or a variant of the same, such as the living recombinant vectors described previously, for example, live bacterial vectors.

Also non-living vectors are suitable for the BASB111 polypeptide, for example "vesicles" or bacterial outer membrane vesicles. ME vesicles are derived from the outer layer of the double-layer membrane of Gram negative bacteria and have been documented in many Gram negative bacteria (Zhou, L et al. 1998. FEMS Microbiol. Lett . 163: 223-228) including C trachomatis and C. psittaci . Also included is a not very exhaustive list of bacterial pathogens that have been reported to produce vesicles: Bordetella pertussis , Borrelia burgdorferi , Brucella melitensis , Brucella ovis , Esherichia coli , Haemophilus influenza , Legionella pneumophila , Moraxella catarrhalis , Neisseria gonorrhoeit , Neisseria gonorrhoeit , Neisseria gonorrhoeit , Neisseria gonorrhoeit , Neisseria gonorrhoeit , Pseudomonas aeruginosa and Yersinia enterocolítica .

Vesicles have the advantage of providing outer membrane proteins in their native conformation and of this mode are particularly useful for vaccines. Vesicles too can be improved for use in vaccines by genetic engineering of bacteria to modify the expression of one or more molecules in the outer membrane In this way, for example, you can enter or regulate by increment (for example, altering the promoter) the expression of a desired immunogenic protein in the membrane external, such as the BASB111 polypeptide. Instead, or in addition, it can regulate by decreasing the expression of molecules in the outer membrane that are not relevant (for example, antigens not immunodominant but variable protectors or proteins) or harmful (for example, toxic molecules such as LPS, or inducers potentials of an immune response). These approaches are discuss in more detail below.

The non-coding flanking regions of the BASB111 gene contain important regulatory elements in the gene expression This regulation takes place both at the level Transcriptional as of translation. The sequence of these regions, before the 5 ′ end and after the 3 ’end of the phase Open gene reading can be obtained by sequencing of DNA This sequence information allows the determination of potential regulatory reasons such as the different elements promoters, terminator sequences, sequence elements inducible, repressors, elements responsible for the variation of phase, the sequence of Shine-Dalgarno, regions with potential secondary structure involved in regulation as well like other types of motifs or regulatory sequences. This Sequence is an additional aspect of the invention.

This sequence information allows the modulation of the natural expression of the BASB111 gene. Regulation by increase in gene expression can be carried out altering the promoter, the sequence of Shine-Dalgarno, repressive elements or operators potentials or any other element involved. Also, the regulation by decreased expression can be carried out by similar modification types. Alternatively, changing the phase variation sequences, gene expression it can be put under the control of the phase variation or it can decouple from this regulation. In another approach, the expression of the gene can be put under the control of one or more elements inducible that allow a regulated expression. The examples of this regulation include, but is not limited to, induction by change of temperature, the addition of inducing substrates such as selected carbohydrates or their derivatives, trace elements, vitamins, cofactors, metal ions, etc.

These modifications, as described above, can be introduced by several different means. The modification of sequences involved in gene expression can be carried out in vivo by random mutagenesis followed by the selection of the desired phenotype. Another approach is to isolate the region of interest and modify it by random mutagenesis or site-directed substitution, insertion or deletion mutagenesis. The modified region can then be reintroduced into the bacterial genome by homologous recombination and the effect on gene expression can be tested. In another approach, knowledge of the sequence of the region of interest can be used to replace or delete all or part of the natural regulatory sequences. In this case, the directed regulatory region is isolated and modified to contain the regulatory elements of another gene, a combination of regulatory elements of different genes, a synthetic regulatory region or any other regulatory region, or deleting selected parts of the regulatory sequence of type wild. Then, these modified sequences can be reintroduced into the bacterium through homologous recombination in the genome. A not very exhaustive list of preferred promoters that could be used for regulation by increased gene expression includes promoters by A, porB, lbpB, tbpB, p111, lst, hpuAB of N. meningitidis or N gonorroheae , ompCD, copB, lbpB , ompE, UspA1; UspA2; TbpB of M. catarrhalis ; p1, p2, p4, p5, p6, lpD, tbpB, D15, Hia, Hmwl, Hmw2 of H. influenzae .

In one example, gene expression can be modulated by exchanging its promoters with a more potent promoter (by isolation of the sequence before the 5 'end of the gene, in vitro modification of this sequence and reintroduction into the genome by homologous recombination). The expression regulated by increase can be obtained both in the bacterium and in the vesicles of the outer membrane released (or made) from the bacteria.

In other examples, the desired approximations can be used to generate recombinant bacterial strains with Enhanced features for vaccine applications. These can be, but not limited to attenuated strains, strains with expression increased number of selected antigens, strains that do not express (or decreased expression) genes that interfere with the immune response, strains with modulated expression of immunodominant proteins, strains with modulated release of outer membrane vesicles.

Thus, it is also provided in the invention a sequence modified above of the BASB111 gene, whose region before the modified 5 'end contains an element heterologous regulator that alters the level of protein expression BASB111 located in the outer membrane. The region before 5 'end according to this aspect of the invention includes the sequence before the 5 'end of the BASB111 gene. The region before the end 5 'begins immediately before the 5' end of the BASB111 gene and normally continues to a position not beyond approximately 1000 bp before the 5 'end of the gene from the codon Startup ATG. In the case of a gene located in a sequence polycistronic (operon), the region before end 5 ’can begin immediately preceding the gene of interest, or preceding the First gene in the operon. Preferably, a region before modified 5 'end according to this aspect of the invention contains a heterologous promoter in a position between 500 and 700 bp before 5 'end of the ATG.

Thus, the invention provides a BASB111 polypeptide, in a modified bacterial vesicle. The invention also provides modified host cells capable of producing non-living membrane vesicle vectors. The invention further provides nucleic acid vectors that comprise the BASB111 gene that has a region before the 5 'end modified that contains a heterologous regulatory element.

Additionally, the invention provides procedures to prepare host cells and bacterial vesicles according to the invention.

This invention provides compositions, particularly vaccine compositions, and procedures that comprise the polypeptides and / or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).

This invention also provides methods using the described polynucleotide, or particular fragments thereof that have been shown to encode non-variable regions of bacterial cell surface proteins, in polynucleotide constructs used in such genetic immunization experiments in animal models of Moraxella infection. catarrhalis These experiments will be particularly useful for identifying protein epitopes capable of eliciting a prophylactic or therapeutic immune response. It is believed that this approach will allow the subsequent preparation of monoclonal antibodies derived from the required organ of the animal that resists or clears an infection successfully, of special value for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Moraxella infection catarrhalis , in mammals, particularly in humans.

\hbox{inmediatamente antes de su uso.}

The invention also includes a vaccine formulation comprising an immunogenic recombinant polypeptide and / or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier. Since polypeptides and polynucleotides can be degraded in the stomach, each is preferably delivered parenterally, including, for example, subcutaneous, intramuscular, intravenous or intradermal administration. Formulations suitable for parenteral administration include sterile aqueous or non-aqueous injection solutions that may contain antioxidants, buffers, bacteriostatic and solute compounds that make the isotonic formulation with the body fluid, preferably with the blood, of the individual; and sterile aqueous and non-aqueous suspensions that may include suspending agents or thickening agents. The formulations can be presented in single-dose or multi-dose containers, for example, sealed ampoules and vials and can be stored under lyophilization conditions that only require the addition of the sterile liquid vehicle

 \ hbox {immediately before use.} 

The vaccine formulation of the invention can also include adjuvant systems to enhance the formulation immunogenicity. Preferably the system of adjuvant preferably causes a TH1 type response.

An immune response can be differentiated in general lines in two extreme categories, which are answer humoral immune or cell-mediated response (characterized traditionally by effector protection mechanisms by antibodies and cell, respectively). These categories of response have been called responses of type TH1 (answer cell-mediated) and TH2 type immune responses (response humoral).

Extreme TH1 type immune responses are can characterize by the generation of specific antigens, haplotype-restricted cytotoxic T lymphocytes and responses of natural killer cells In mice, TH1 type responses are often characterized by the generation of subtype antibodies IgG2a, while in humans these correspond to antibodies of type IgG1. TH2 type immune responses are characterized by generation of a wide range of immunoglobulin isotypes including in IgG1, IgA and IgM mice.

It can be considered that the driving force that there is behind the development of these two types of immune response are the cytokines High levels of TH1 type cytokines tend to favor the induction of immune responses mediated by cells against a certain antigen while levels elevated cytokines of type TH2 tend to favor induction of humoral immune responses against the antigen.

The distinction between TH1 and TH2 type immune responses is not absolute. In reality, an individual will support an immune response that is described as predominantly TH1 or predominantly TH2. However, it is often convenient to consider the cytokine families in the terms described by Mosmann and Coffman for murine CD4 + T cell clones ( Mosmann, TR and Coffman, RL (1989) TH1 and TH2 cells different patterns of lymphokine secretion lead to different functional properties Annual Review of lmmunology, 7, p. 145-173 ). Traditionally, TH1 type responses are associated with the production by T lymphocytes of the INF-? And IL-2 cytokines. Other cytokines often directly associated with the induction of TH1 type immune responses are not produced by T cells, such as IL-2. In contrast, TH2 type responses are associated with the secretion of IL-4, IL-5, IL-6 and IL-13.

It is known that certain vaccine adjuvants are particularly suitable for the stimulation of cytokine responses of type TH1 or TH2. Traditionally, the best indicators of the TH1: TH2 balance of the immune response after vaccination or infection include the direct measurement of the production of TH1 or TH2 cytokines by in vitro T lymphocytes after antimimulation and / or proportion measurement IgG1: IgG2a of antigen specific antibody responses.

Thus, a TH1 type adjuvant is one that preferably stimulates isolated T cell populations to produce elevated levels of TH1 type cytokines when in vitro stimulated with antigen and promotes the development of both CD8 + cytotoxic T lymphocyte responses and immunoglobulin responses. antigen specific, associated with the TH1 type isotype.

In International Patent Application No. WO 94/00153 and WO 95/17209 are described adjuvants that are capable of preferably stimulate the TH1 cell response.

One of these adjuvants is the monophosphoryl lipid A 3-O-deacetylated (3D-MPL). It is known from the GB document 2220211 (Ribi). Chemically it is a mixture of monophosphoryl lipid A 3-O-deacetylated with chains 4, 5 or 6 acetylated and is manufactured by Ribi Immunochem, Montana. A preferred form of monophosphoryl lipid A 3-O-deacetylated is described in the European Patent 0 689 454 B1 (SmithKline Beecham Biologicals S.A.).

Preferably, the particles of 3D-MPL are small enough to be sterilized by filtration through a 0.22 membrane micrometers (European Patent Number 0 689 454). He 3D-MPL will be presented in the range of 10 \ mug-100 \ mug, preferably 25-50 µg per dose in which the antigen is will typically present in a range of 2-50 \ mug per dose.

Another preferred adjuvant comprises QS21, a non-toxic fraction purified by HPLC derived from the crust of Quillaja saponaria Molina. Optionally this can be mixed with monophosphoryl-lipid A 3-O-deacylated (3D-MPL), optionally together with a vehicle.

The production procedure of QS21 is described in US Pat. No. 5,057,540.

They have been previously described (document WO96 / 33739) formulations of non-reactive adjuvants which They contain QS21. It has been proven that these formulations that comprise QS21 and cholesterol are TH1 stimulating adjuvants that they are successful when formulated together with an antigen.

Additional adjuvants that stimulate preferably the TH1 cell response include immunomodulatory oligonucleotides, for example, non-CpG sequences methylated as described in WO 96/02555.

It is also contemplated that combinations of different TH1 stimulating adjuvants, such as those mentioned earlier in this document, they provide an adjuvant that is a preferential stimulator of TH1 cell response. For example, QS21 can be formulated together with 3D-MPL. The QS21 ratio: 3D-MPL will typically be order from 1: 10 to 10: 1; preferably from 1: 5 to 5: 1 and often substantially 1: 1. The preferred range of optimal synergy is from 2.5: 1 to 1: 1 3D-MPL: QS21.

Preferably a vehicle is also present in the vaccine composition according to the invention. The vehicle can be an oil-in-water emulsion or an aluminum salt, such as aluminum phosphate or hydroxide of
aluminum.

An emulsion of preferred oil-in-water comprises a metabolizable oil, such as squalene, alpha tocopherol and Tween 80. In a particularly preferred aspect the antigens in the Vaccine composition according to the invention is combined with QS21 and 3D-MPL in this emulsion. Additionally, the Oil-in-water emulsion may contain span 85 and / or lecithin and / or Tricapriline

Typically for administration to humans, QS21 and 3D-MPL will be presented in a vaccine in the 1 \ mug-200 \ mug range, such as 10-100 \ mug, preferably 10 \ mug-50 \ mug per dose. Typically, the emulsion of oil in water will comprise 2 to 10% squalene, 2 to 10% of alpha tocopherol and 0.3 to 3% Tween 80. Preferably the ratio of squalene: alpha tocopherol is equal to or less than 1 since This provides a more stable emulsion. Span 85 can also be present at a level of 1%. In some cases, it may be advantageous that the vaccines of the present invention contain a additional stabilizer

Non-toxic emulsions of oil in water preferably contain a non-toxic oil, for example, squalane or squalene and an emulsifier, for example, Tween 80, in an aqueous vehicle The aqueous vehicle can be, for example, phosphate buffered saline.

WO 95/17210 describes a especially potent adjuvant formulation involving QS21, 3D-MPL and tocopherolin, in an oil emulsion in Water.

The present invention also provides a polyvalent vaccine composition comprising a formulation for vaccine of the invention in combination with other antigens, in particular antigens useful for the treatment of cancers, autoimmune diseases and related ailments. This composition for polyvalent vaccine may include a TH1 inducing adjuvant as described earlier in this document.

While the invention has been described with Reference to certain BASB111 polypeptides and polynucleotides, should be understand that this covers fragments of the polypeptides or natural polynucleotides, and polypeptides and polynucleotides similar with additions, deletions or substitutions that do not affect substantially to the immunogenic properties of the polypeptides or recombinant polynucleotides.

Compositions, kits and administration

In a further aspect of the invention there are provided compositions comprising a polynucleotide
BASB111 and / or a BASB111 polypeptide for administration to a cell or a multicellular organism.

The invention also relates to compositions comprising a polynucleotide and / or a polypeptide discussed in This document or its agonists or antagonists. Polypeptides and polynucleotides of the invention can be used in combination with a sterile or non-sterile vehicle or vehicles for use with cells, tissues organisms, such as a pharmaceutical vehicle suitable for administration to an individual. These compositions they comprise, for example, an additive to the medium or an amount therapeutically effective of a polypeptide and / or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient. These vehicles may include, but are not limited to, solution saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation must conform to the mode of administration. The invention also relates to packages and kits Diagnostics and pharmaceuticals comprising one or more containers filled with one or more of the ingredients of the compositions of the invention mentioned above.

Polypeptides, polynucleotides and other compounds of the invention separately or together with others compounds, such as therapeutic compounds.

       \ newpage
    

Pharmaceutical compositions can be administered in any convenient and effective way including, for example, administration topically, orally, anally, vaginally, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal, among others.

The active agent can be administered to a individual, in therapy or as a prophylactic, as a composition injectable, for example, as a sterile aqueous dispersion, preferably isotonic.

In a further aspect, the present invention provides pharmaceutical compositions comprising an amount therapeutically effective of a polypeptide and / or polynucleotide, such as the soluble form of a polypeptide and / or polynucleotic of the present invention, agonist or antagonist peptide or compound of small molecule, in combination with a vehicle or excipient pharmaceutically acceptable. These vehicles include, but without limitations, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The invention also refers to pharmaceutical packages and kits comprising one or more containers loaded with one or more of the ingredients of the compositions of the invention mentioned above. Can be employ polypeptides, polynucleotides and other compounds of the present invention separately or together with other compounds, such as therapeutic compounds

The composition will adapt to the path of administration, for example, by a systemic or oral route. The Preferred forms of systemic administration include injection, typically by intravenous injection. Other ones can be used injection routes, such as subcutaneous, intramuscular or intraperitoneal. Alternative means for systemic administration include transmucosal and transdermal administration using agents penetration such as bile salts, fusidic acid or other detergents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric formulation or encapsulated, oral administration may also be possible. The administration of these compounds may also be topical and / or located, in the form of ointment, pastes, gels, solutions, powders and Similar.

For administration to mammals, and particularly to humans, the dosage level is expected Daily active agent will be 0.01 mg / kg to 10 mg / kg, typically about 1 mg / kg The doctor in any case will determine the actual dose that will be the most appropriate for an individual and will vary with the age, weight and response of the particular individual. The Previous dosages are examples of an average case. May there are, of course, individual cases that deserve intervals of higher or lower dosage, and these are within the scope of the invention.

The dosage interval required depends on the choice of the peptide, the route of administration, the nature of the formulation, the nature of the ailment of the subject and the assessment of the attending physician. The right dosages, however, they are in the range of 0.1-100 / mug / kg of subject's body weight.

A vaccine composition is conveniently In injectable form. Conventional adjuvants can be used to Boost the immune response. A dose unit suitable for vaccination is 0.5-5 micrograms / kg of antigen, and this dose is preferably administered in 1-3 times and in an interval of 1-3 weeks. With the dose range indicated, no toxicological effects will be observed adverse effects with the compounds of the invention which could prevent their Administration to suitable individuals.

Wide variations are expected, however the necessary dosage, in view of the diversity of compounds available and the different efficiencies of various routes of administration. For example, the administration could be expected oral need dosages higher than those of the administration by intravenous injection. Variations in these Dosage levels can be adjusted using empirical routines conventional for optimization, as is well understood in the technique.

Sequence databases, sequences in a tangible medium and algorithms

\hbox{el paquete de
programas GCG.}

The polynucleotide and polypeptide sequences constitute a valuable source of information with which to determine their bi-dimensional and three-dimensional structures, as well as to identify additional sequences of similar homology. These approaches more easily favor the storage of the sequence in a computer-readable medium and then use the data stored in a program of known macromolecular structure or to search a sequence database using well-known search tools, such how

 \ hbox {the package of
GCG programs.} 

The invention also provides the procedures for the analysis of sequences or chains of characters, especially genetic sequences or sequences of encoded protein Preferred analysis procedures of sequence include, for example, analysis procedures of sequence homology, such as identity analysis and similarity, analysis of the structure of DNA, RNA and protein, sequence assembly, cladistic analysis, motive analysis sequencing, determination of open reading frames, analysis of nucleic acid signals, codon usage analysis, nucleic acid clipping analysis and peak analysis of sequencing chromatogram

A procedure based on computer techniques to perform homology identification. This procedure comprises the steps of: providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium; Y comparing said first polynucleotide sequence with at least one second polynucleotide or polypeptide sequence to identify homologies

A procedure based on computer techniques to perform homology identifications, said procedure comprising the steps of: providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium; Y comparing said first polypeptide sequence with at least one second polynucleotide or polypeptide sequence to identify homologies

Definitions

"Identity," as is known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by sequence comparison. In the art, "identity" also means the degree of sequence relationship between polypeptide or polynucleotide sequences, as the case may be, as determined by the coincidence between the chains of these sequences. "Identity" can be easily calculated by known procedures, including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing Informatics and Genome Projects , Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data , Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology , von Heine, G., Academic Press, 1987; and Sequence Analysis Primer , Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math ., 48: 1073 (1988). Procedures are designed to determine the identity to achieve the greatest coincidence between the sequences analyzed. In addition, the procedures for determining identity are collected in publicly available computer programs. Computer program procedures for determining the identity between two sequences include, but are not limited to, the GAP program of the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1) : 387 (1984)) , BLASTP, BLASTN (Altschul, SF et al., J. Molec. Biol. 215: 403-410 (1990)), and FASTA (Pearson and Lipman Proc. Natl. Acad. Sci. USA 85 : 2444-2448 (1988 )). The BLAST family of programs is publicly available from the NCBI and other sources ( BLAST Manual, Altschul , S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215 : 403-410 (1990) The well-known Smith Waterman algorithm can also be used to determine identity.

The parameters for sequence comparison Polypeptides include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 by Henikoff and Henikoff, Proc. Natl Acad. Sci. USA. 89: 10915-10919 (1992)

Penalty for hole: 8

Penalty for gap length: 2

A useful program with these parameters is publicly available as the "hollow" Genetics program Computer Group, Madison WI. The parameters mentioned above are the default parameters for peptide comparisons (together with no penalties for gaps in the ends).

The parameters for the comparison of Polynucleotides include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = +10, no match = 0

Penalty for hole: 50

Penalty for gap length: 3

Available as: The "hollow" program of Genetics Computer Group, Madison WI. These are the parameters by defect for nucleic acid comparisons.

A preferred meaning for "identity" of polynucleotides and polypeptides, as appropriate, are provided to continued in (1) and (2).

(1) Embodiments of polynucleotides include furthermore an isolated polynucleotide comprising a sequence polynucleotide that has at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity with the reference sequence of the SEQ ID No. 1, wherein said polynucleotide sequence can be identical to the reference sequence of SEQ ID No. 1: or may include up to a certain integer number of nucleotide alterations compared to the reference sequence, in which said alterations are selected from the group consisting of at least a deletion, substitution, including transition and transversion, or nucleotide insertion, and in which said alterations can occur in the 5 ’or 3’ terminal positions of the sequence of reference nucleotides or anywhere between those positions terminals, interleaved individually between the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and in which said number of alterations nucleotide is determined by multiplying the total number of nucleotides of SEQ ID No. 1 by the integer that defines the percentage of identity divided by 100 and then subtracting that product of said total number of nucleotides in SEQ ID No. 1, or:

n_ {n} \ leq x_ {n} - (x_ {n} \ cdot Y),

where n_ {n} is the number of nucleotide alterations, x_ {n} is the total number of nucleotides of SEQ ID No. 1, and is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100% and \ cdot is the symbol for the multiplication operator, and where any non-integer product of x_ {n} e and rounded down to the nearest previous integer to subtract it from x_ {n}. The alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID No. 2 can create antisense mutations, of wrong direction or offset reading frame in this coding sequence and therefore alter the polypeptide encoded by the polynucleotide after these alterations

As an example, a polynucleotide sequence of the present invention may be identical to the sequence of reference of SEQ ID No. 1, which may be 100% identical, or it can include up to a certain whole number of alterations of nucleic acids compared to the reference sequence of so the percentage of identity is less than 100%. These alterations are selected from the group consisting of at least a deletion, substitution, including transition and transversion, or nucleic acid insertion, and in which said alterations can occur in the 5 'or 3' terminal positions of the sequence reference polynucleotide or anywhere between those terminal positions, interleaved individually between nucleotides of the reference sequence or in one or more groups contiguous within the reference sequence. The number of nucleic acid alterations for a percentage of identity given is determined by multiplying the total number of nucleic acids of SEQ ID No. 1 for the integer that defines the percentage of identity, divided by 100 and then subtracting that product of said total number of nucleic acids of SEQ ID NO. 1, or:

n_ {n} \ leq x_ {n} - (x_ {n} \ cdot Y),

where n_ {n} is the number of nucleic acid alterations, x_ {n} is the total number of nucleic acids of SEQ ID No. 1, and is, for example, 0.70 for the 70%, 0.80 for 80%, 0.85 for 85%, etc. .. \ cdot is the symbol for the multiplication operator, and where any product does not integer of x_ {n} e and rounded down to the previous integer closest to subtract it from x_ {n}.

(2) Polypeptide embodiments include furthermore an isolated polypeptide comprising a polypeptide having at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity with a reference sequence of the polypeptide of SEQ ID No. 2, in the that said polypeptide sequence may be identical to the sequence of reference of SEQ ID No. 2 or may include up to a certain integer number of amino acid alterations compared to the reference sequence, in which said alterations are select from the group consisting of at least one deletion, substitution, including conservative and non-conservative substitution, or insertion of nucleic acid, and in which said alterations terminal amino or carboxyl positions of the reference polypeptide sequence or anywhere between those terminal positions, sandwiched individually between nucleic acids in the reference sequence or in one or more groups contiguous within the reference sequence, and in which said number of amino acid alterations is determined by multiplying the total number of amino acids of SEQ ID No. 2 for the integer that define the percentage of identity divided by 100 and then subtracting that product from said total number of amino acids in the ID SEC No. 2, or:

n_ {a} \ leq x_ {a} - (x_ {a} \ cdot Y),

where n_ {a} is the number of amino acid alterations, x_ {a} is the total number of amino acids of SEQ ID NO. 2, and is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100% and \ cdot is the symbol for the multiplication operator, and where any non-integer product of x_ {a} e and rounded down to the nearest previous integer to subtract it from for}.

As an example, a polypeptide sequence of the present invention may be identical to the sequence of reference of SEQ ID No. 2, which may be 100% identical, or it can include up to a certain whole number of alterations of amino acids compared to the reference sequence so that the percentage of identity is less than 100% identity. These alterations are selected from the group consisting of at minus one deletion, substitution, including conservative substitution and non-conservative, or amino acid insertion, and in which said alterations can occur in the amino or carboxyl positions terminals of the reference polypeptide sequence or in anywhere between those terminal positions, interspersed individually between the amino acids of the reference sequence or in one or more contiguous groups within the sequence of reference. The number of amino acid alterations for a given identity percentage is determined by multiplying the number total amino acids of SEQ ID No. 2 by the integer that defines the identity percentage divided by 100 and then subtracting that product from said total number of amino acids from the ID SEC No. 2, or:

n_ {a} \ leq x_ {a} - (x_ {a} \ cdot Y),

where n_ {a} is the number of amino acid alterations, x_ {a} is the total number of amino acids of SEQ ID No. 2, and is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, etc. and \ cdot is the symbol for the multiplication operator, and where any product does not integer from x_ {a} and rounded down to the previous integer closest to subtract it from for}.

"Individual (or individuals)", when used in this document in reference to an organism, means a eukaryotic multicellular, which includes, but is not limited to, a metazoan, a mammal, an eager, a bovid, an ape, a primate and a human.

"Isolated" means altered "by hand of man "from his natural state, that is, if he produced in nature, has been changed or removed from its environment original, or both. For example, a polynucleotide or a polypeptide present in nature in a living organism is not "isolated", but the same polynucleotide or separate polypeptide of the coexisting materials of its natural state is "isolated", as the term is used in this document. In addition, a polynucleotide or polypeptide that is introduced into a organism by transformation, genetic manipulation or by any other recombination procedure is "isolated" even if it is still present in that organism, it may be organism be alive or not alive.

"Polynucleotide (or polynucleotides)" is generally refers to any polyiribonucleotide or polyideoxyriblonucleotide, which can be RNA or DNA without modify or modified RNA or DNA, including chain regions single or double.

"Variant" refers to a polynucleotide or polypeptide that differs from a polynucleotide or polypeptide of reference, but that retains the essential properties. A typical variant of a polynucleotide differs in sequence nucleotide of another reference polynucleotide. The changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes can give place for substitutions, additions, deletions, mergers and amino acid truncation in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs from another polypeptide of reference in the amino acid sequence. Generally, the differences are limited so that the polypeptide sequences reference and variant are very similar overall identical in many regions, identical. A variant and a reference polypeptide may differ in the sequence of amino acids in one or more substitutions, additions or deletions in any combination A substituted or inserted amino acid residue It may or may not be encoded by the genetic code. A variant of a polynucleotide or polypeptide can be natural, as a variant allelic, or it can be a variant that is not known in nature. Unnatural variants of polynucleotides and polypeptides can be done by mutagenesis techniques or by synthesis direct.

"Disease (or diseases)" means any disease caused by, or related to, an infection by a bacterium, including, for example, otitis media in infants and in children, pneumonia in the elderly, sinusitis, nosocomial infections and invasive diseases, chronic otitis media with loss of hearing, accumulation of fluid in the middle ear, nerve damage auditory, delayed speech learning, infection in the upper respiratory tract and inflammation of the middle ear.

Examples

The following examples were performed using conventional techniques, which are well known and routine for those skilled in the art, except where otherwise described in detail. The examples are illustrative, and do not limit the invention.

Example one

Discovery and confirmatory sequencing of the DNA of the BASB111 gene of the ATCC 43617 strain of Moraxella catarrhalis

The DNA sequence of the BASB111 gene of the ATCC 43617 strain of Moraxella catarrhalis (also referred to as strain MC2931) is shown in SEQ ID No. 1. Translation of the BASB111 polynucleotide sequence is shown in SEQ ID No. 2.

Example 2

Construction of the plasmid to express BASB111 recombinant A: BASB111 cloning

The restriction sites of Eco RI and Sal I introduced by genetic engineering in the sense amplification primers MC-Lip2-Fn / t-RI (5 '- AGG CAG AGG GAA TTC ATG AAT TTT GGT AAA ATT AAT GG-3') [SEQ ID No. 3] and complementary MC-Lip2RCh / t-SaI (5'- AGG CAG AGG GTC GAC TTA ATG GTG ATG GTG ATG GTG CCA GCC TTT GAT AAC ACC ATC TT - 3 ') [SEC ID No. 4], respectively, allowed directional cloning of a PCR product in the E. coli pTLZ2 expression plasmid so that a mature BASB111 protein could be expressed as a fusion protein containing an affinity chromatography tag (His) 6 at the end C-terminal The BASB111 PCR product was purified from the amplification reaction using gel-based centrifugal column (QiaGen) according to the manufacturers instructions. To produce the required Eco RI and Sal I ends, necessary for cloning, the purified PCR products were digested sequentially until termination with the Eco RI and Salt I restriction enzymes as recommended by the manufacturer (Life Technologies). After the first restriction digestion, the PCR product was purified by a centrifugal column as before to remove salts and eluted in sterile water before the second enzymatic digestion. The digested DNA fragment was purified again using silica gel based centrifugal columns before ligation with plasmid pTLZ2.

B: Production of the expression vector

To prepare the expression plasmid pTLZ2 for ligation, it was digested to termination in a similar manner with both Eco RI and Sal I and then treated with a calf intestine phosphatase (FIT. Sim0.02 units / pmoles of end 5 ', Life Technologies) as directed by the manufacturer to prevent self-ligation. Approximately 5 times a molar excess of the digested fragment was used with respect to the vector prepared to schedule the ligation reaction. A ligation reaction was performed in \20 µm (1616 ° C, 1616 hours), using methods well known in the art using T4 DNA ligase (22.0 units / reaction, Life Technologies). A linkage aliquot (55 µl) was used to transform electrocompetent JM109 cells according to procedures well known in the art. After an overcrowding period of ~ 2-3 hours at 37 ° C in ~ 1.0 ml of LB culture, the transformed cells were placed on LB agar plates containing ampicillin (100 µg / ml). Antibiotics were included in the selection. The plates were incubated overnight at 37 ° C for ~ 16 hours. The individual ApR colonies were chopped with sterile sticks and used to inoculate in freshly prepared LB Apr plates as "patches" as well as ~ 1.9 ml of LB.ApR culture medium. Both patched plates and culture medium were incubated overnight at 37 ° C in a conventional incubator (plates) or in a shaking water bath. A full cell based PCR analysis was used to verify that the transformants contained the insertion of BASB111 DNA. Here, the ,01.0 ml of LB Ap culture medium overnight was transferred to a 1.5 ml polypropylene tube and the cells were collected by centrifugation in a Beckmann microcentrifuge (sim3 min, room temperature, 1212 .000xg). The cell pellet was resuspended in? 200 µl of sterile water and an aliquot of? 10? Was used to schedule a PCR reaction in a final volume of? 50 ml containing both amplification primers, sense and complementary, of BASB111 . The final concentrations of the components of the PCR reaction were essentially the same as those specified in Example 2 except that ,05.0 Taq polymerase units were used. The initial stage of denaturation at 95 ° C was increased to 3 minutes to ensure the thermal breakdown of bacterial cells and the release of plasmid DNA. An ABI model 9700 thermocycler and 32 cycles of a three-stage thermal amplification profile was used, ie 95 ° C, 45 sec; 55-58 ° C, 45 sec, 72 ° C, 1 min, to amplify the BASB111 fragment of the lysate samples of the transformants. After thermal amplification, an aliquot of the sim20 µL reaction was analyzed by agarose gel electrophoresis (0.8% agarose in a Tris-acetate-EDTA buffer (TAE)). The DNA fragments were visualized after gel electrophoresis by UV illumination and ethidium bromide staining. A DNA molecular size pattern (1 Kb ladder, Life Technlogies) was electrophoresed in parallel with the test samples and used to estimate the size of the PCR products. The transformants that produced the expected size PCR product were identified as strains containing an expression construct of BASB111. Next, the inducible expression of recombinant BASB111 in strains containing expression plasmids was analyzed.

C: Analysis of the expression of transformants PCR-positive

For each positive transformant in PCR identified above,? 5.0 ml of LB medium was inoculated containing ampicillin (100 µg / ml) with plaque cells in patch and grown overnight at 37 ° C with shaking (? 250 rpm). An aliquot of the crop sown from a night (~ 1.0 ml) in a 125 ml Erlenmeyer flask containing sim25 of LB Ap culture medium and cultured at 37 ° C with stirring (? 250 rpm) until crop turbidity reached a D.O. 600 of sim0.5, that is, semi-logarithmic phase (normally approximately 1.5-2.0 hours). At this time, Approximately half of the culture (~ 12.5 ml) was transferred to a second 125 ml flask and protein expression was induced Recombinant BASB111 adding IPTG (1.0 M backup solution prepared in sterile water, Sigma) at a final concentration of 1.0 mM. Incubation of both IPTG-induced and non-culture induced was continued for? 4 more hours at 37 ° C with stirring.  After the induction period, samples were withdrawn (~ 1.0 ml) both induced and uninduced culture and the cells are collected by centrifugation in a microcentrifuge at temperature ambient for \3 minutes. Cell sediments individual were resuspended in \ sim50 \ mul of sterile water, then they were mixed with an equal volume of sample buffer of Laemlli SDS-PAGE x2 containing 2-mercaptoethanol and placed in a water bath boiling for sim3 min to denature the proteins. Equal volumes (\ sim15 \ mul) of crude oils from both lysates IPTG-induced and non-induced cells were loaded on a gel in duplicate of 12% polyacrylamide in Tris / glycine (Minigels of 1 mm thick, Novex). Induced lysate samples and not induced underwent electrophoresis along with markers of pretended molecular weight (SeeBlue, Novex) under conditions conventional using an SDS / Tris / glycine development buffer conventional (BioRad). After electrophoresis, a gel was stained with bright blue of Coomassie R250 (BioRad) and then it dyed to visualize the new or new BASB111 proteins inducible with IPTG. The second gel was subjected to electrotransfer to a PVDF membrane (pore size of 0.45 micrometers, Novex) for sim2 hours at 4 ° C using an apparatus of Mini-Protean II transfer from BioRad and buffer Methanol transfer (20%) of Towbin. Membrane block and incubations with the antibody were performed according to procedures well known in the art. An antibody was used monoclonal anti-RGS (His) 3, followed by a second rabbit anti-mouse conjugate antibody with HRP (QiaGen), to confirm the expression and identity of the BASB111 recombinant protein. The pattern display of anti-His antibody reactivity was reached using an ABT insoluble substrate or using Hyperfilm with the system of ECL chemiluminescence from Amersham.

Example 4

Production of recombinant BASB111 Bacterial strain

A recombinant expression strain of E. coli JM109 containing a plasmid (pTLZ2) encoding BASB111 from M. catarrhalis was used to produce cell mass for the purification of the recombinant protein. The expression strain was grown on LB agar plates containing 100 µg / ml ampicillin ("Ap") to ensure that pTLZ2 was maintained. For cryopreservation at -80 ° C, the strain was propagated in LB medium containing the same concentration of antibiotics and then mixed with an equal amount of LB medium containing 30% glycerol (w / v).

Media

The fermentation media used for Recombinant protein production were composed of YT medium 2x (Difco) containing Ap at 100 µg / ml. It was added to the medium to the antifoam fermenter at 0.25 ml / l (Antifoam 204, Sigma). To induce recombinant protein expression, it was added to the IPTG fermenter (Isopropil β-D-Thiogalactopyranoside) (1 mM, final).

Fermentation

An Erlenmeyer 500 culture flask was inoculated ml, which contained a working volume of 50 ml, with 0.3 ml of Frozen and thawed culture quickly or with several colonies of a selective culture on agar plates, and incubated for approximately 12 hours at 37 ± 1 ° C on a platform with stirring at 150 rpm (Innova 2100, New Brunswich Scientific). This seeded culture was then used to inoculate a fermenter 5 l of work volume containing YT 2x medium and both antibiotics Ap. The fermenter (Bioflo 3000, New Brunswick Scientific) was handled at 37 ± 1 ° C, 0.2-0.4 VVM of Induced air, 250 rpm on Rushton paddle wheels. Not controlled pH neither in the flask sowing culture nor in the fermenter. During fermentation, the pH ranged from 6.5 to 7.3 in the fermenter IPTG (1.0 M stock solution, prepared in sterile water) to the fermenter when the crop reached semilogarithmic growth (sim0.7 units of D.O. 600). The cells were induced for 2-4 hours and at then, they were collected by high speed centrifugation using a 28RS Heraeus (Sepatech) or RC5C (Sorvall centrifuge) Instruments) The cell paste was stored at -20 until processing

Purification Chemicals and materials

Imidazole, guanidinium hydrochloride, Tris (hydroxymethyl) and EDTA (ethylene diamine tetraacetic acid) grade biotechnological or higher, all were obtained from Ameresco Chemical, Solon, Ohio. Triton X-100 (t-Octylphenoxypolyethoxy ethanol), Triton X-114, sodium phosphate, monobasic and Urea were reagent grade or higher and were obtained from Sigma Chemical Company, St. Louis, Missouri. Glacial acetic acid and acid hydrochloric were obtained from Mallincrodt Baker Inc., Phillipsburg, New Jersey. Methanol was obtained from Fisher Scientific, Fairlawn, New Jersey. Pefabloc © SC (4 (2-aminoethyl) -benzenesulfonylfluoride), the complete protease inhibitor mixture tablets and PMSF (phenylmethyl sulfonylfluoride) were obtained from Roche Diagnostics Corporation, Indianapolis, Indiana. Bestatin, Pepstatin A and protease inhibitor E-64 se obtained from Calbiochem, La Jolla, California. Saline solution Buffered with Dulbecco phosphate (1xPBS) was obtained from Quality Biological, Inc, Gaithersburg, Maryland. Saline solution buffered with Dulbecco phosphate (10xPBS) were obtained from BioWhittaker, Walkersville, Maryland. Antibody Penta-His without BSA was obtained from QiaGen, Valencia, California. The goat IgG anti-mouse AffiniPure peroxidase conjugate was obtained from Jackson Immuno Research, West Grove, Penn. AEC's unique solution was obtained from Zymed, South of San Francisco, California All these chemicals were grade reactive or higher

The high flow nickel chelating Sepharose resin was obtained from Pharmacia, Sweden, California. Prefabricated 4-10% and 10-20% Tris-Glycine polyacrylamide gels, all buffers and development solutions, SeeBlue pretended patterns, multi-colored MultiMark patterns and PVDF transfer membranes were obtained from Novex, San Diego, California Silver staining kits from SDS-PAGE were obtained from Daiichi Pure Chemicals Company Limited, Tokyo, Japan. The Coomassie staining solution was obtained from Bio-Rad Laboratories, Hercules, California. 0.2 m Acrodisc © PF syringe filters were obtained from Pall Gelman Sciences, Ann Arbor, Michigan. The 25 mm GD / X disposable syringe filters were obtained from Whatman Inc. Clifton, New Jersey. The 8,000 PMPC dialysis tube was obtained from BioDesing Inc, Od New York, Carmal New York. BCA protein assay reagents and the 3,500 PMPC Snake Skin dialysis tube were obtained from Pierce Chemical Co., Rockford, Illinois.

Purification of recombinant BASB111 from E. coli Extraction and purification

The cell paste was thawed at temperature ambient for 30 to 60 minutes. After breaking up the cell paste, the supernatant was partitioned with ice-cold PBS containing Triton X-114 at 1%.

The cells were removed, the extract was heated at 37 ° C and the phases were distributed by centrifugation.

This fraction was then passed through a balanced high flow nickel Chelating Sepharose resin in PBS (pH 7.5) containing 10% glycerol and Triton X100 at 0.05% The protein was eluted with the same buffer it contained. 200 mM Imidazole. Fractions containing the eluted protein, concentrated in a cell with agitation of 10 kDa cut-off point and then dialyzed against PBS containing 0.1% Triton X100.

As shown in Figure 1-A, purified BASB111 protein appeared in analysis by SDS-PAGE as a double migration to approximately 36 kDa (estimated relative molecular mass). It was estimated that purity It was greater than 90%. Both bands reacted against an antibody mouse monoclonal obtained against the subject 6-Histidine (Figure 1-B).

Biochemical characterizations Analysis by SDS-PAGE and immunoblot

The purified recombinant BASB111 protein is separated in 4-20% polyacrylamide gels and electrophoretically transferred to PVDF membranes at 100 V for 1 time as previously described (Thebaine et al., Proc. Natl. Acad. Sci. USA 76: 4350-4354). Then the membranes were pretreated with 25 ml of saline buffered with Dulbecco phosphate containing 5% skimmed milk powder. All subsequent incubations were performed using this buffer. Pretreatment

PVDF membranes were incubated with 25 ml of a 1: 500 dilution of preimmune serum or rabbit immune serum anti-His for 1 hour at room temperature. TO then the PVDF membranes were washed twice with buffer wash (20 mM Tris buffer, pH 7.5, containing 150 sodium chloride mM and 0.05% Tween-20). PVDF membranes are incubated with 25 ml of a 1: 5000 dilution of goat IgG against peroxidase-labeled rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 minutes at room temperature. TO then the PVDF membranes were washed 4 times with buffer washed and developed with 3-amino-9-ethylcarbazole and urea peroxide supplied by Zymed (San Francisco, CA) for 10 minutes each.

Example 5

Production of antisera against recombinant BASB111

The multipurpose antisera directed against the BASB111 protein was generated by vaccinating two or four rabbits with the purified recombinant BASB111 protein. Each animal receives a total of three subcutaneous immunizations of approximately 10 mg of BASB111 protein per injection at intervals of approximately 21 days The animals bled before the first immunization ("pre-bleeding") and on days 49 and 56.

The multipurpose antisera directed against the BASB111 protein was generated by vaccinating six mice with the protein BASB111 purified recombinant. Each animal receives a total of two or three subcutaneous immunizations of approximately 10 mg of BASB111 protein by injection at intervals of approximately 14 days. The animals bled before the first immunization ("presangrado") and one week after the last immunization.

BASB111 anti-protein values were measured by an ELISA using recombinant BASB111 protein purified (4 µg / well). The value is defined as the values of the midpoint calculated using the logistic model of 4 parameters using the KL Fit software. The values obtained after the immunization for the sera of rabbits and mice was 1: 270000 and 1: 46500, respectively. The antibodies were used as the first antibody to identify the protein in a type transfer Western, as described in example 7 below. The Western blot shows the presence of an antibody anti-BASB111 in sera from immunized animals (figure 2).

Example 6

Immunological characterization: Exposed surface of BASB111

BASB111 anti-protein values were determined by an ELISA using formalin-killed whole cells of strains 14, 358, 216, 2926 of Moraxella catarrhalis (20 µg / well). The value is defined as the midpoint values calculated using the 4-parameter logistic model using SoftMax Pro software.

The values observed with rabbit immune sera (1: 2600, 1: 2300, 1: .430, 1: 3300 respectively) demonstrate that BASB111 protein is detected on the surface of M. catarrhalis cells.

Example 7

Immunological characterization: analysis by immunoblot

M. catarrhalis ATCC 43617 was cultured on Muller Hinton agar plates for 24 hours at 36 ° C. Several colonies were used to inoculate 50 ml of BHI culture medium in a 250 ml flask. The cultures were grown for approximately 5 hours at 200 rpm until A 620 was approximately 0.6 and collected by centrifugation. The cells were then concentrated tenfold and the equivalent of 4x10 8 CFU was solubilized in 150 ml of PAGE sample buffer (360 mM Tris buffer, pH 8.8 containing 4% sodium dodecyl sulfate and glycerol at 20%) and the suspension was incubated at 100 ° C for 5 minutes. The solubilized cells were separated on 4-20% polyacrylamide gels and the separated proteins were electrophoretically transferred to 100 V nitrocellulose membranes for 1 hour as previously described (Thebaine et al., 1979, Proc. Natl. Acad. Sci. USA 76: 4350-4354). Next, the nitrocellulose membranes were pretreated with 50 ml of Dulbecco phosphate buffered saline solution containing 3% bovine serum albumin (BSA). All subsequent incubations were performed using this pretreatment buffer.

Nitrocellulose membranes were incubated with 250 µl of a 1:20 dilution of preimmune serum or serum Immune mouse for 2 hours at room temperature. TO then the nitrocellulose membranes were washed three times with wash buffer (20 mM Tris buffer, pH 7.5 containing chloride 150 mM sodium and 0.05% Tween-20), the membranes of nitrocellulose were incubated with 50 ml of a 1: 500 dilution of Ig of biotin-marked anti-mouse goat (Amersham Life Sciences Products) for 60 minutes at room temperature. TO then the nitrocellulose membranes were washed 3 times with wash buffer and incubated with 50 ml of a 1: 1000 dilution of streptavidin peroxidase (Amersham) for 60 minutes at room temperature. Next, the membranes of nitrocellulose was washed 3 times with wash buffer and developed with 4-chloro-1-naphthol supplied by Sigma for ten minutes each.

In the ATCC 43617 strain of Moraxella , a protein of approximately 29 kDa was detected (corresponding to the expected molecular weight of BASB111) that is reactive with the antisera (Figure 3).

Example 8

Immunological characterization: bactericidal activity

Completely mediated cytotoxic activity of anti-BASB111 antibodies was examined to determine the potential as a vaccine for the antiserum against BASB111 protein prepared as described above. The activities of preimmune serum and anti-BASB111 antiserum were examined in death of complement-mediated M. catarrhalis .

The strains of M. catarrhalis were grown in Mueller Hinton plates for 24 hours at 36 ° C. Several colonies were added to 15 ml of BHI in the 125 ml flasks. The cultures were grown for 4 hours at 200 rpm until A620 = 0.4. After a washing step, the sediment was resuspended in HBSS and the strain was diluted to obtain 28,500 CFU per milliliter.

50 µl of preimmune sera and anti-BASB111 sera (inactivated at 56 ° C for 30 minutes) were deposited in the first well of a 96-well plate and in the other wells of the same line 1/2 dilutions were deposited in HBSS . Subsequently, 25 µl of diluted live M. catarrhalis was added and the mixture was incubated for 15 minutes at room temperature. In each well, rabbit breeding complement (Pel Freez, Clinical Systems, Brown Deer, WI, USA) was added to a dilution of work defined in advance in a toxicity test.

The microplates were covered and incubated for 1 hour at 37 ° C at 200 rpm.

Each trial included a complement control (serum-free wells containing sources of active complement or inactivated), a positive control (wells containing serum with a known value of bactericidal antibodies), a control of culture (wells without complement).

The bactericidal value of rabbit antiserum or mice (death of 50% of the homologous strain) was <1:60 (preimmune) and> 1: 316 (immune).

Deposited materials

A deposit containing a strain of Moraxella catarrhalis Catlin was deposited with the American Type Culture Collection (in this document "ATCC") on June 21, 1997 and assigned deposit number 43617. The deposit was described as Branhamella catarrhalis ( Frosck and Kolle) and is a lyophilized library with an insertion of 1.5-2., 9 kb built from an isolate of M. catarrhalis obtained from a transtracheal aspirate of a coal miner with chronic bronchitis. The deposit is described in Antimicrob. Agents Chemother 21: 506-508 (1982).

The deposit of the Moraxella catarrhalis strain is referred to herein as "the deposited strain" or as "the DNA of the deposited strain".

The deposited strain contains a BASB111 gene of full length

A deposit of the pMC-ORF1 / 2 vector consisting of Moraxella catarrhalis DNA inserted into pQE30 was deposited with the American Type Culture Collection (ATCC) on February 12, 1999 and assigned deposit number 207118.

The sequences of the polynucleotides contained in the deposited strain or clone, as well as the amino acid sequence of any polypeptide encoded by these, is decisive in the case of any conflict with any description of the sequences of this document.

The deposit of the deposited strains was made in the terms of the Budapest Treaty on Recognition International Deposit of Microorganisms for the purpose of Patent Procedures The deposited strains will be, so irrevocable and without restriction or conditions, publicly available for subject matter experts and are not an acknowledgment that requires a deposit for the permit, as set out in the U.S. 35. \ NAK112.

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Sequence Information

Polynucleotide and polypeptide sequences BASB111

SEQ ID No. 1

BASB111 polynucleotide sequence of Moraxella catarrhalis strain ATCC43617

2

SEC ID No. 2

Sequence of the BASB111 polypeptide of Moraxella catarrhalis deduced from the polynucleotide of the

SEQ ID No. 1

3

SEC ID No. 3

AGG CAG AGG GAA TTC ATG AAT TTT GGT AAA ATT AAT GG

SEC ID No. 4

AGG CAG AGG GTC GAC TTA ATG GTG ATG GTG ATG GTG CCA GCC TTT GAT AAC ACC ATC TT

<110> SmithKline Beecham S. A.

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<120> New compounds

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<130> BM45395

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<160> 4

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<170> FastSEQ for Windows, Version 3.0

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<210> 1

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<211> 831

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<212> DNA

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<213> Moraxella catarrhalis

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<400> 1

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4

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<210> 2

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<211> 276

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<212> PROT

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<213> Moraxella catarrhalis

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<400> 2

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5

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<210> 3

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<211> 38

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<212> DNA

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<213> Artificial sequence

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<220>

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<223> Primer

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<400> 3

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\hskip-.1em\dddseqskip

aggacagaggg aattcatgaa ttttggtaaa attaatgg

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38

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aggacagaggg aattcatgaa ttttggtaaa attaatgg

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38

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<210> 4

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<211> 59

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<212> DNA

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<213> Artificial sequence

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<220>

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<223> Primer

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<400> 4

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aggcagaggg tcgacttaat ggtgatggtg atggtgccag cctttgataa caccatctt

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59

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aggcagaggg tcgacttaat ggtgatggtg atggtgccag cctttgataa caccatctt

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59

Claims (28)

1. An isolated polypeptide comprising a amino acid sequence that has at least 85% identity with the amino acid sequence of SEQ ID No. 2 over length full of SEQ ID No. 2. 2. An isolated polypeptide according to claim 1 in which the amino acid sequence has at least 95% of amino acid sequence identity of SEQ ID NO. 2. 3. The polypeptide according to claim 1 which It comprises the amino acid sequence of SEQ ID NO. 2. 4. An isolated polypeptide of SEQ ID NO. 2. 5. An immunogenic fragment comprising a amino acid sequence that has at least 20 contiguous amino acids of the amino acid sequence of SEQ ID No. 2, whose fragment (if it is necessary when you join a vehicle) it is capable of originating a immune response that recognizes the polypeptide of SEQ ID NO. 2. 6. A polypeptide or an immunogenic fragment according to any one of claims 1 to 5 wherein said polypeptide or said immunogenic fragment is part of a protein of major fusion. 7. An isolated polynucleotide encoding a polypeptide or an immunogenic fragment according to any of the claims 1 to 6. 8. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide that has at minus 85% identity with the amino acid sequence of the ID SEC No. 2 over the full length of SEQ ID No. 2, or a nucleotide sequence complementary to said polynucleotide isolated. 9. An isolated polynucleotide comprising a nucleotide sequence that has at least 85% identity with a nucleotide sequence encoding a polypeptide of SEQ ID No. 2 over the entire coding region, or a sequence nucleotide complementary to said isolated polynucleotide. 10. An isolated polynucleotide comprising a nucleotide sequence that has at least 85% identity with that of SEQ ID No. 1 over the full length of SEQ ID No. 1, or a nucleotide sequence complementary to said polynucleotide isolated. 11. The isolated polynucleotide according to a any one of claims 7 to 10 wherein the identity is at least 95% with SEQ ID No. 1. 12. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO. 2, or the immunogenic fragment of claim 5 or 6. 13. An isolated polynucleotide comprising the polynucleotide of SEQ ID No. 1. 14. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO. 2, obtainable by selecting an appropriate library in conditions stringent hybridization with a labeled probe that has the sequence of SEQ ID No. 1 or a fragment thereof. 15. An expression vector or a microorganism recombinant live comprising an isolated polynucleotide according to a any of claims 7-14. 16. A recombinant living microorganism that It comprises an expression vector according to claim 15. 17. A host cell comprising the expression vector of claim 15. 18. A host cell membrane according to claim 17 expressing an isolated polypeptide that comprises an amino acid sequence that has at least 85% of identity with the amino acid sequence of SEQ ID NO. 2. 19. A method for producing a polypeptide or an immunogenic fragment of claims 1 to 6 which comprises culturing a host cell of claim 17 under conditions sufficient for the production of said polypeptide or said immunogenic fragment and recovering the polypeptide from the medium of
culture. 20. A procedure to express a polynucleotide of any one of the claims 7-14 which comprises transforming a cell host with an expression vector comprising at least one of said polynucleotides and culturing said host cell in sufficient conditions for the expression of any one of said polynucleotides. 21. A vaccine composition comprising a effective amount of the polypeptide or the immunogenic fragment of a any one of claims 1 to 6 and a vehicle pharmaceutically acceptable. 22. A vaccine composition comprising a effective amount of the polynucleotide of any one of the claims 7 to 14 and a pharmaceutically carrier acceptable. 23. The vaccine composition according to one of claim 21 or 22 wherein said composition comprises at least one other Moraxella catarrhalis antigen. 24. An antibody generated against the polypeptide or immunogenic fragment according to any one of the claims 1 to 6. 25. A method for diagnosing a Moraxella infection, which comprises identifying a polypeptide or an immunological fragment according to any one of claims 1-6 or an antibody that is immunospecific for said polypeptide, present in a biological sample of an animal of which You suspect you have this infection. 26. Use of a composition comprising a immunologically effective amount of a polypeptide or fragment immunogenic according to any one of the claims 1-6 in the preparation of a medicine for use in the generation of an immune response in an animal. 27. Use of a composition comprising a immunologically effective amount of a polynucleotide according to a any of claims 7-14 in the preparation of a medicine for use in generating a immune response in an animal. 28. A therapeutic composition useful for the treatment of humans suffering from Moraxella catarrhalis comprising at least one antibody directed against the polypeptide or immunogenic fragment of claims 1-6 and a suitable pharmaceutical carrier.